Artificial nucleic acid molecules for improved protein expression

ABSTRACT

The invention relates to an artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal. The invention further relates to a vector comprising the artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal, to a cell comprising the artificial nucleic acid molecule or the vector, to a pharmaceutical composition comprising the artificial nucleic acid molecule or the vector and to a kit comprising the artificial nucleic acid molecule, the vector and/or the pharmaceutical composition. The invention also relates to a method for increasing protein production from an artificial nucleic acid molecule and to the use of a 3′-UTR for a method for increasing protein production from an artificial nucleic acid molecule. Moreover, the invention concerns the use of the artificial nucleic acid molecule, the vector, the kit or the pharmaceutical composition as a medicament, as a vaccine or in gene therapy.

This application is divisional of U.S. application Ser. No. 15/534,496,filed Jun. 9, 2017, which is a national phase application under 35U.S.C. § 371 of International Application No. PCT/EP2015/002501, filedDec. 11, 2015, which claims the benefit of International Application No.PCT/EP2014/003334, filed Dec. 12, 2014, each of which is incorporatedherein by reference in its entirety.

This invention was made with Government support under Agreement No.HR0011-11-3-0001 awarded by DARPA. The Government has certain rights inthe invention.

FIELD OF THE INVENTION

The invention relates to an artificial nucleic acid molecule comprisingan open reading frame and a 3′-UTR comprising at least one poly(A)sequence or a polyadenylation signal. The invention further relates to avector comprising the artificial nucleic acid molecule comprising anopen reading frame and a 3′-UTR comprising at least one poly(A) sequenceor a polyadenylation signal, to a cell comprising the artificial nucleicacid molecule or the vector, to a pharmaceutical composition comprisingthe artificial nucleic acid molecule or the vector and to a kitcomprising the artificial nucleic acid molecule, the vector and/or thepharmaceutical composition. The invention also relates to a method forincreasing protein production from an artificial nucleic acid moleculeand to the use of a 3′-UTR for a method for increasing proteinproduction from an artificial nucleic acid molecule. Moreover, theinvention concerns the use of the artificial nucleic acid molecule, thevector, the kit or the pharmaceutical composition as a medicament, as avaccine or in gene therapy.

BACKGROUND OF THE INVENTION

Gene therapy and genetic vaccination belong to the most promising andquickly developing methods of modern medicine. They may provide highlyspecific and individual options for therapy of a large variety ofdiseases. Particularly, inherited genetic diseases but also autoimmunediseases, cancerous or tumour-related diseases as well as inflammatorydiseases may be the subject of such treatment approaches. Also, it isenvisaged to prevent (early) onset of such diseases by these approaches.

The main conceptual rational behind gene therapy is appropriatemodulation of impaired gene expression associated with pathologicalconditions of specific diseases. Pathologically altered gene expressionmay result in lack or overproduction of essential gene products, forexample, signalling factors such as hormones, housekeeping factors,metabolic enzymes, structural proteins or the like. Altered geneexpression may not only be due to mis-regulation of transcription and/ortranslation, but also due to mutations within the ORF coding for aparticular protein. Pathological mutations may be caused by e.g.chromosomal aberration, or by more specific mutations, such as point orframe-shift-mutations, all of them resulting in limited functionalityand, potentially, total loss of function of the gene product. However,misregulation of transcription or translation may also occur, ifmutations affect genes encoding proteins, which are involved in thetranscriptional or translational machinery of the cell. Such mutationsmay lead to pathological up- or down-regulation of genes, which are—assuch—functional. Genes encoding gene products which exert suchregulating functions, may be, e.g., transcription factors, signalreceptors, messenger proteins or the like. However, loss of function ofsuch genes encoding regulatory proteins may, under certaincircumstances, be reversed by artificial introduction of other factorsacting further downstream of the impaired gene product. Such genedefects may also be compensated by gene therapy via substitution of theaffected gene itself.

Genetic vaccination allows to evoke a desired immune response toselected antigens, such as characteristic components of bacterialsurfaces, viral particles, tumour antigens or the like. Generally,vaccination is one of the pivotal achievements of modern medicine.However, effective vaccines are currently available only for a smallernumber of diseases. Accordingly, infections that are not preventable byvaccination still affect millions of people every year.

Commonly, vaccines may be subdivided into “first”, “second” and “third”generation vaccines. “First generation” vaccines are, typically,whole-organism vaccines. They are based on either live and attenuated orkilled pathogens, e.g. viruses, bacteria or the like. The major drawbackof live and attenuated vaccines is the risk for a reversion tolife-threatening variants. Thus, although attenuated, such pathogens maystill intrinsically bear unpredictable risks. Killed pathogens may notbe as effective as desired for generating a specific immune response. Inorder to minimize these risks, “second generation” vaccines weredeveloped. These are, typically, subunit vaccines, consisting of definedantigens or recombinant protein components, which are derived frompathogens.

Genetic vaccines, i.e. vaccines for genetic vaccination, are usuallyunderstood as “third generation” vaccines. They are typically composedof genetically engineered nucleic acid molecules, which allow expressionof peptide or protein (antigen) fragments characteristic for a pathogenor a tumor antigen in vivo. Genetic vaccines are expressed uponadministration to a patient and uptake by competent cells. Expression ofthe administered nucleic acids results in production of the encodedproteins. In the event these proteins are recognized as foreign by thepatient's immune system, an immune response is triggered.

As can be seen from the above, both methods, gene therapy and geneticvaccination, are essentially based on the administration of nucleic acidmolecules to a patient and subsequent transcription and/or translationof the encoded genetic information. Alternatively, genetic vaccinationor gene therapy may also comprise methods, which include isolation ofspecific body cells from a patient to be treated, subsequent in vitrotransfection of such cells, and re-administration of the treated cellsto the patient.

DNA as well as RNA may be used as nucleic acid molecules foradministration in the context of gene therapy or genetic vaccination.DNA is known to be relatively stable and easy to handle. However, theuse of DNA bears the risk of undesired insertion of the administeredDNA-fragments into the patient's genome potentially resulting in loss offunction of the impaired genes. As a further risk, the undesiredgeneration of anti-DNA antibodies has emerged. Another drawback is thelimited expression level of the encoded peptide or protein that isachievable upon DNA administration and its transcription/translation.Among other reasons, the expression level of the administered DNA willbe dependent on the presence of specific transcription factors, whichregulate DNA transcription. In the absence of such factors, DNAtranscription will not yield satisfying amounts of RNA. As a result, thelevel of translated peptide or protein obtained is limited.

By using RNA instead of DNA for gene therapy or genetic vaccination, therisk of undesired genomic integration and generation of anti-DNAantibodies is minimized or avoided. However, RNA is considered to be arather unstable molecular species which may readily be degraded byubiquitous RNAses.

In vivo, RNA-degradation contributes to the regulation of the RNAhalf-life time. That effect was considered and proven to fine tune theregulation of eukaryotic gene expression (Friedel et al., Conservedprinciples of mammalian transcriptional regulation revealed by RNAhalf-life, Nucleic Acid Research, 2009, 1-12). Accordingly, eachnaturally occurring mRNA has its individual half-life depending on thegene from which the mRNA is derived. It contributes to the regulation ofthe expression level of this gene. Unstable RNAs are important torealize transient gene expression at distinct points in time. However,long-lived RNAs may be associated with accumulation of distinct proteinsor continuous expression of genes. In vivo, the half life of mRNAs mayalso be dependent on environmental factors, such as hormonal treatment,as has been shown, e.g., for insulin-like growth factor I, actin, andalbumin mRNA (Johnson et al., Newly synthesized RNA: Simultaneousmeasurement in intact cells of transcription rates and RNA stability ofinsulin-like growth factor I, actin, and albumin in growthhormone-stimulated hepatocytes, Proc. Natl. Acad. Sci., Vol. 88, pp.5287-5291, 1991).

For gene therapy and genetic vaccination, usually stable RNA is desired.This is, on the one hand, due to the fact that the product encoded bythe RNA-sequence shall accumulate in vivo. On the other hand, the RNAhas to maintain its structural and functional integrity when preparedfor a suitable dosage form, in the course of its storage, and whenadministered. Thus, considerable attention was dedicated to providestable RNA molecules for gene therapy or genetic vaccination in order toprevent them from being subject to early degradation or decay.

It has been reported that the G/C-content of nucleic acid molecules mayinfluence their stability. Thus, nucleic acids comprising an increasedamount of guanine (G) and/or cytosine (C) residues may be functionallymore stable than nucleic acids containing a large amount of adenine (A)and thymine (T) or uracil (U) nucleotides. In this context, WO02/098443provides a pharmaceutical composition containing an mRNA that isstabilised by sequence modifications in the translated region. Such asequence modification takes advantage of the degeneracy of the geneticcode. Accordingly, codons which contain a less favourable combination ofnucleotides (less favourable in terms of RNA stability) may besubstituted by alternative codons without altering the encoded aminoacid sequence. This method of RNA stabilization is limited by theprovisions of the specific nucleotide sequence of each single RNAmolecule which is not allowed to leave the space of the desired aminoacid sequence. Also, that approach is restricted to coding regions ofthe RNA.

As an alternative option for mRNA stabilisation, it has been found thatnaturally occurring eukaryotic mRNA molecules contain characteristicstabilising elements. For example, they may comprise so-calleduntranslated regions (UTR) at their 5′-end (5′UTR) and/or at their3′-end (3′UTR) as well as other structural features, such as a 5′-capstructure or a 3′-poly(A) tail. Both, 5′UTR and 3′UTR are typicallytranscribed from the genomic DNA and are, thus, an element of thepremature mRNA. Characteristic structural features of mature mRNA, suchas the 5′-cap and the 3′-poly(A) tail (also called poly(A) tail orpoly(A) sequence) are usually added to the transcribed (premature) mRNAduring mRNA processing.

A 3′-poly(A) tail is typically a monotonous sequence stretch of adeninenucleotides, which is enzymatically added to the 3′-end of the nascentmRNA. Typically, the poly(A) tail of a mammalian mRNA contains about 250adenine nucleotides. It was found that the length of such a 3′-poly(A)tail is a potentially critical element for the stability of theindividual mRNA. In this context, Holtkamp et al. reported that apoly(A) tail consisting of 120 nucleotides resulted in a more stablemRNA molecule, which was expressed more efficiently, than a shorterpoly(A) tail (Holtkamp et al., Modification of antigen-encoding RNAincreases stability, translational efficacy, and T-cell stimulatorycapacity of dendritic cells, Blood, Vol. 108, pp. 4009-4017, 2006).However, according to Holtkamp et al., a further extension of thepoly(A) tail does not lead to an additional increase in mRNA stabilityor expression. It was further reported that enzymatic adenylation of anmRNA comprising a poly(A) tail further enhances expression of the mRNAafter electroporation into T cells (Zhao et al., Multiple Injections ofElectroporated Autologous T Cells Expressing a Chimeric Antigen ReceptorMediate Regression of Human Disseminated Tumor, Caner Res., Vol. 70(22),pp. 9053-9061, 2010).

Nearly all eukaryotic mRNAs end with a poly(A) sequence that is added totheir 3′-end by the ubiquitous cleavage/polyadenylation machinery. Thepresence of a poly(A) sequence at the 3′-end is one of the mostrecognizable features of eukaryotic mRNAs. After cleavage, mostpre-mRNAs, with the exception of replication-dependent histonetranscripts, acquire a polyadenylated tail. In this context, 3′-endprocessing is a nuclear co-transcriptional process that promotestransport of mRNAs from the nucleus to the cytoplasm and affects thestability and the translation of mRNAs. Formation of this 3′ end occursin a two step reaction directed by the cleavage/polyadenylationmachinery and de-pends on the presence of two sequence elements in mRNAprecursors (pre-mRNAs); a highly conserved hexanucleotide AAUAAA(polyadenylation signal) and a downstream G/U-rich sequence. In a firststep, pre-mRNAs are cleaved between these two elements. In a second steptightly coupled to the first step the newly formed 3′ end is extended byaddition of a poly(A) sequence consisting of 200-250 adenylates whichaffects subsequently all aspects of mRNA metabolism, including mRNAexport, stability and translation (Dominski, Z. and W. F. Marzluff(2007), Gene 396(2): 373-90.).

The only known exception to this rule are the replication-dependenthistone mRNAs, which terminate with a histone stem-loop instead of apoly(A) sequence. Exemplary histone stem-loop sequences are described inLopez et al. (Davila Lopez, M., & Samuelsson, T. (2008), RNA (New York,N.Y.), 14(1), 1-10. doi:10.1261/rna.782308.).

The stem-loops in histone pre-mRNAs are typically followed by apurine-rich sequence known as the histone downstream element (HDE).These pre-mRNAs are processed in the nucleus by a single endonucleolyticcleavage approximately 5 nucleotides downstream of the stem-loop,catalyzed by the U7 snRNP through base pairing of the U7 snRNA with theHDE.

Due to the requirement to package newly synthesized DNA into chromatin,histone synthesis is regulated in concert with the cell cycle. Increasedsynthesis of histone proteins during S phase is achieved bytranscriptional activation of histone genes as well aspost-transcriptional regulation of histone mRNA levels. It could beshown that the histone stem-loop is essential for allposttranscriptional steps of histone expression regulation. It isnecessary for efficient processing, export of the mRNA into thecytoplasm, loading onto polyribosomes, and regulation of mRNA stability.

In the above context, a 32 kDa protein was identified, which isassociated with the histone stem-loop at the 3′-end of the histonemessages in both the nucleus and the cytoplasm. The expression level ofthis stem-loop binding protein (SLBP) is cell cycle regulated and ishighest during S-phase when histone mRNA levels are increased. SLBP isnecessary for efficient 3′-end processing of histone pre-mRNA by the U7snRNP. After completion of processing, SLBP remains associated with thestem-loop at the end of mature histone mRNAs and stimulates theirtranslation into histone proteins in the cytoplasm. (Dominski, Z. and W.F. Marzluff (2007), Gene 396(2): 373-90). Interestingly, the RNA bindingdomain of SLBP is conserved throughout metazoa and protozoa (DavilaLopez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308) and it could be shown that its binding to thehistone stem-loop sequence is dependent on the stem-loop structure andthat the minimum binding site contains at least 3 nucleotides 5′ and 2nucleotides 3′ of the stem-loop (Pandey, N. B., et al. (1994), Molecularand Cellular Biology, 14(3), 1709-1720 and Williams, A. S., & Marzluff,W. F., (1995), Nucleic Acids Research, 23(4), 654-662.).

Even though histone genes are generally classified as either“replication-dependent”, giving rise to mRNA ending in a histonestem-loop, or “replacement-type”, giving rise to mRNA bearing apoly(A)-tail instead, naturally occurring mRNAs containing both ahistone stem-loop and poly(A) or oligo(A) 3′ thereof have beenidentified in some very rare cases. Sanchez et al. examined the effectof naturally occurring oligo(A) tails appended 3′ of the histonestem-loop of histone mRNA during Xenopus oogenesis using luciferase as areporter protein and found that the oligo(A) tail is an active part ofthe translation repression mechanism that silences histone mRNA duringoogenesis and its removal is part of the mechanism that activatestranslation of histone mRNAs (Sanchez, R. and W. F. Marzluff (2004), MolCell Biol 24(6): 2513-25).

Furthermore, the requirements for regulation of replication dependenthistones at the level of pre-mRNA processing and mRNA stability havebeen investigated using artificial constructs coding for the markerprotein alpha globin, taking advantage of the fact that the globin genecontains introns as opposed to the intron-less histone genes. For thispurpose, constructs were generated in which the alpha globin codingsequence was followed by a histone stem-loop signal (histone stem-loopfollowed by the histone downstream element) and a polyadenylation signal(Whitelaw, E., et al. (1986). Nucleic Acids Research, 14(17), 7059-7070;Pandey, N. B., & Marzluff, W. F. (1987). Molecular and Cellular Biology,7(12), 4557-4559; Pandey, N. B., et al. (1990). Nucleic Acids Research,18(11), 3161-3170).

Also, it was shown that the 3′UTR of α-globin mRNA may be an importantfactor for the well-known stability of α-globin mRNA (Rodgers et al.,Regulated α-globin mRNA decay is a cytoplasmic event proceeding through3′-to-5′ exosome-dependent decapping, RNA, 8, pp. 1526-1537, 2002). The3′UTR of α-globin mRNA is obviously involved in the formation of aspecific ribonucleoprotein-complex, the α-complex, whose presencecorrelates with mRNA stability in vitro (Wang et al., An mRNA stabilitycomplex functions with poly(A)-binding protein to stabilize mRNA invitro, Molecular and Cellular Biology, Vol 19, No. 7, July 1999, p.4552-4560).

Irrespective of factors influencing mRNA stability, effectivetranslation of the administered nucleic acid molecules by the targetcells or tissue is crucial for any approach using nucleic acid moleculesfor gene therapy or genetic vaccination. Along with the regulation ofstability, also translation of the majority of mRNAs is regulated bystructural features like UTRs, 5′-cap and 3′-poly(A) tail. In thiscontext, it has been reported that the length of the poly(A) tail mayplay an important role for translational efficiency as well. Stabilizing3′-elements, however, may also have an attenuating effect ontranslation.

Further regulative elements, which may have an influence on expressionlevels, may be found in the 5′UTR. For example, it has been reportedthat synthesis of particular proteins, e.g. proteins belonging to thetranslational apparatus, may be regulated not only at thetranscriptional but also at the translational level. For example,translation of proteins encoded by so called ‘TOP-genes’ may bedown-regulated by translational repression. Therein, the term ‘TOP-gene’relates to a gene corresponding to an mRNA that is characterized by thepresence of a TOP sequence at the 5′end and in most cases by agrowth-associated translation regulation (ladevaia et al., Alltranslation elongation factors and the e, f, and h subunits oftranslation initiation factor 3 are encoded by 5′-terminaloligopyrimidine (TOP) mRNAs; RNA, 2008, 14:1730-1736). In this context,a TOP sequence—also called the ‘5′-terminal oligopyrimidinetract’—typically consists of a C residue at the cap site, followed by anuninterrupted sequence of up to 13 or even more pyrimidines (Avni etal., Vertebrate mRNAs with a 5′-terminal pyrimidine tract are Candidatesfor translational repression in quiescent cells: characterization of thetranslational cis-regulatory element, Molecular and Cellular Biology,1994, p. 3822-3833). These TOP sequences are reported to be present inmany mRNAs encoding components of the translational machinery and to beresponsible for selective repression of the translation of these TOPcontaining mRNAs due to growth arrest (Meyuhas, et al., TranslationalControl of Ribosomal Protein mRNAs in Eukaryotes, Translational Control.Cold Spring Harbor Monograph Archive. Cold Spring Harbor LaboratoryPress, 1996, p. 363-388).

It is the object of the invention to provide artificial nucleic acidmolecules, which may be suitable for use as a medicament or a vaccine,preferably for application in gene therapy and/or genetic vaccination.Particularly, it is the object of the invention to provide artificialnucleic acid molecules, such as an mRNA species, which provide forimproved protein production from said artificial nucleic acid molecules.Another object of the present invention is to provide nucleic acidmolecules encoding such a superior mRNA species, which may be amenablefor use as a medicament or a vaccine, preferably in gene therapy and/orgenetic vaccination. It is a further object of the present invention toprovide a pharmaceutical composition, preferably for use as a medicamentor a vaccine, preferably in gene therapy and/or genetic vaccination. Insummary, it is the object of the present invention to provide improvednucleic acid species, which overcome the above discussed disadvantagesof the prior art by means of a cost-effective and straightforwardapproach.

The object underlying the present invention is solved by the claimedsubject-matter.

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Adaptive immune response: The adaptive immune response is typicallyunderstood to be an antigen-specific response of the immune system.Antigen specificity allows for the generation of responses that aretailored to specific pathogens or pathogen-infected cells. The abilityto mount these tailored responses is usually maintained in the body by“memory cells”. Should a pathogen infect the body more than once, thesespecific memory cells are used to quickly eliminate it. In this context,the first step of an adaptive immune response is the activation of naïveantigen-specific T cells or different immune cells able to induce anantigen-specific immune response by antigen-presenting cells. Thisoccurs in the lymphoid tissues and organs, through which naïve T cellsare constantly passing. The three cell types that may serve asantigen-presenting cells are dendritic cells, macrophages, and B cells.Each of these cells has a distinct function in eliciting immuneresponses. Dendritic cells may take up antigens by phagocytosis andmacropinocytosis and may become stimulated by contact with e.g. aforeign antigen to migrate to the local lymphoid tissue, where theydifferentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents or other appropriate stimuli to express MHC molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may also be important to induce T cells. MHC-moleculesare, typically, responsible for presentation of an antigen to T cells.Therein, presenting the antigen on MHC molecules leads to activation ofT cells, which induces their proliferation and differentiation intoarmed effector T cells. The most important function of effector T cellsis the killing of infected cells by CD8+ cytotoxic T cells and theactivation of macrophages by Th1 cells, which together make upcell-mediated immunity, and the activation of B cells by both Th2 andTh1 cells to produce different classes of antibody, thus driving thehumoral immune response. T cells recognize an antigen by their T cellreceptors, which do not recognize and bind the antigen directly, butinstead recognize short peptide fragments, e.g. of pathogen-derivedprotein antigens, e.g. so-called epitopes, which are bound to MHCmolecules on the surfaces of other cells.

Adaptive immune system: The adaptive immune system is essentiallydedicated to eliminate or prevent pathogenic growth. It typicallyregulates the adaptive immune response by providing the vertebrateimmune system with the ability to recognize and remember specificpathogens (to generate immunity), and to mount stronger attacks eachtime the pathogen is encountered. The system is highly adaptable becauseof somatic hypermutation (a process of accelerated somatic mutations),and V(D)J recombination (an irreversible genetic recombination ofantigen receptor gene segments). This mechanism allows a small number ofgenes to generate a vast number of different antigen receptors, whichare then uniquely expressed on each individual lymphocyte. Because thegene rearrangement leads to an irreversible change in the DNA of eachcell, all of the progeny (offspring) of such a cell will then inheritgenes encoding the same receptor specificity, including the Memory Bcells and Memory T cells that are the keys to long-lived specificimmunity.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in thebroadest sense is typically a pharmacological and/or immunological agentthat may modify, e.g. enhance, the effect of other agents, such as adrug or vaccine. It is to be interpreted in a broad sense and refers toa broad spectrum of substances. Typically, these substances are able toincrease the immunogenicity of antigens. For example, adjuvants may berecognized by the innate immune systems and, e.g., may elicit an innateimmune response. “Adjuvants” typically do not elicit an adaptive immuneresponse. Insofar, “adjuvants” do not qualify as antigens. Their mode ofaction is distinct from the effects triggered by antigens resulting inan adaptive immune response.

Antigen: In the context of the present invention, “antigen” referstypically to a substance which may be recognized by the immune system,preferably by the adaptive immune system, and is capable of triggeringan antigen-specific immune response, e.g. by formation of antibodiesand/or antigen-specific T cells as part of an adaptive immune response.Typically, an antigen may be or may comprise a peptide or protein, whichmay be presented by the MHC to T-cells. In the sense of the presentinvention, an antigen may be the product of translation of a providednucleic acid molecule, preferably an mRNA, as defined herein. In thiscontext, also fragments, variants and derivatives of peptides andproteins comprising at least one epitope are understood as antigens. Inthe context of the present invention, tumour antigens and pathogenicantigens as defined herein are particularly preferred.

Artificial nucleic acid molecule: An artificial nucleic acid moleculemay typically be understood to be a nucleic acid molecule, e.g. a DNA oran RNA, that does not occur naturally. In other words, an artificialnucleic acid molecule may be understood as a non-natural nucleic acidmolecule. Such nucleic acid molecule may be non-natural due to itsindividual sequence (which does not occur naturally) and/or due to othermodifications, e.g. structural modifications of nucleotides which do notoccur naturally. An artificial nucleic acid molecule may be a DNAmolecule, an RNA molecule or a hybrid-molecule comprising DNA and RNAportions. Typically, artificial nucleic acid molecules may be designedand/or generated by genetic engineering methods to correspond to adesired artificial sequence of nucleotides (heterologous sequence). Inthis context, an artificial sequence is usually a sequence that may notoccur naturally, i.e. it differs from the wild type sequence by at leastone nucleotide. The term “wild type” may be understood as a sequenceoccurring in nature. Further, the term “artificial nucleic acidmolecule” is not restricted to mean “one single molecule” but is,typically, understood to comprise an ensemble of identical molecules.Accordingly, it may relate to a plurality of identical moleculescontained in an aliquot.

Bicistronic RNA, multicistronic RNA: A bicistronic or multicistronic RNAis typically an RNA, preferably an mRNA, that may typically have two(bicistronic) or more (multicistronic) open reading frames (ORF). Anopen reading frame in this context is a sequence of codons that istranslatable into a peptide or protein.

Carrier/polymeric carrier: A carrier in the context of the invention maytypically be a compound that facilitates transport and/or complexationof another compound (cargo). A polymeric carrier is typically a carrierthat is formed of a polymer. A carrier may be associated to its cargo bycovalent or non-covalent interaction. A carrier may transport nucleicacids, e.g. RNA or DNA, to target cells. The carrier may—for someembodiments—be a cationic component.

Cationic component: The term “cationic component” typically refers to acharged molecule, which is positively charged (cation) at a pH valuetypically from 1 to 9, preferably at a pH value of or below 9 (e.g. from5 to 9), of or below 8 (e.g. from 5 to 8), of or below 7 (e.g. from 5 to7), most preferably at a physiological pH, e.g. from 7.3 to 7.4.Accordingly, a cationic component may be any positively charged compoundor polymer, preferably a cationic peptide or protein, which ispositively charged under physiological conditions, particularly underphysiological conditions in vivo. A “cationic peptide or protein” maycontain at least one positively charged amino acid, or more than onepositively charged amino acid, e.g. selected from Arg, His, Lys or Orn.Accordingly, “polycationic” components are also within the scopeexhibiting more than one positive charge under the conditions given.

5′-cap: A 5′-cap is an entity, typically a modified nucleotide entity,which generally “caps” the 5′-end of a mature mRNA. A 5′-cap maytypically be formed by a modified nucleotide, particularly by aderivative of a guanine nucleotide. Preferably, the 5′-cap is linked tothe 5′-terminus via a 5′-5′-triphosphate linkage. A 5′-cap may bemethylated, e.g. m7GpppN, wherein N is the terminal 5′ nucleotide of thenucleic acid carrying the 5′-cap, typically the 5′-end of an RNA.Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′-phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety.

Cellular immunity/cellular immune response: Cellular immunity typicallyrelates to the activation of macrophages, natural killer cells (NK),antigen-specific cytotoxic T-lymphocytes, and the release of variouscytokines in response to an antigen. In more general terms, cellularimmunity is not based on antibodies, but on the activation of cells ofthe immune system. Typically, a cellular immune response may becharacterized e.g. by activating antigen-specific cytotoxicT-lymphocytes that are able to induce apoptosis in cells, e.g. specificimmune cells like dendritic cells or other cells, displaying epitopes offoreign antigens on their surface. Such cells may be virus-infected orinfected with intracellular bacteria, or cancer cells displaying tumorantigens. Further characteristics may be activation of macrophages andnatural killer cells, enabling them to destroy pathogens, andstimulation of cells to secrete a variety of cytokines that influencethe function of other cells involved in adaptive immune responses andinnate immune responses.

DNA: DNA is the usual abbreviation for deoxy-ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually deoxy-adenosine-monophosphate,deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate anddeoxy-cytidine-monophosphate monomers, which are—by themselves—composedof a sugar moiety (deoxyribose), a base moiety and a phosphate moiety,and polymerise to form a characteristic backbone structure. The backbonestructure is, typically, formed by phosphodiester bonds between thesugar moiety of the nucleotide, i.e. deoxyribose, of a first and aphosphate moiety of a second, adjacent monomer. The specific order ofthe monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the DNA sequence. DNA may be singlestranded or double stranded. In the double stranded form, thenucleotides of the first strand typically hybridize with the nucleotidesof the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.

Epitope: Epitopes (also called “antigen determinants”) can bedistinguished in T cell epitopes and B cell epitopes. T cell epitopes orparts of the proteins in the context of the present invention maycomprise fragments preferably having a length of about 6 to about 20 oreven more amino acids, e.g. fragments as processed and presented by MHCclass I molecules, preferably having a length of about 8 to about 10amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), orfragments as processed and presented by MHC class II molecules,preferably having a length of about 13 or more amino acids, e.g. 13, 14,15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragmentsmay be selected from any part of the amino acid sequence. Thesefragments are typically recognized by T cells in the form of a complexconsisting of the peptide fragment and an MHC molecule, i.e. thefragments are typically not recognized in their native form. B cellepitopes are typically fragments located on the outer surface of(native) protein or peptide antigens as defined herein, preferablyhaving 5 to 15 amino acids, more preferably having 5 to 12 amino acids,even more preferably having 6 to 9 amino acids, which may be recognizedby antibodies, i.e. in their native form. Such epitopes of proteins orpeptides may furthermore be selected from any of the herein mentionedvariants of such proteins or peptides. In this context, antigenicdeterminants can be conformational or discontinuous epitopes, which arecomposed of segments of the proteins or peptides as defined herein thatare discontinuous in the amino acid sequence of the proteins or peptidesas defined herein, but are brought together in the three-dimensionalstructure or continuous or linear epitopes, which are composed of asingle polypeptide chain.

Fragment of a sequence: A fragment of a sequence may typically be ashorter portion of a full-length sequence of e.g. a nucleic acidmolecule or an amino acid sequence. Accordingly, a fragment typicallyconsists of a sequence that is identical to the corresponding stretchwithin the full-length sequence. A preferred fragment of a sequence inthe context of the present invention, consists of a continuous stretchof entities, such as nucleotides or amino acids corresponding to acontinuous stretch of entities in the molecule the fragment is derivedfrom, which represents at least 5%, 10%, 20%, preferably at least 30%,more preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, and mostpreferably at least 80% of the total (i.e. full-length) molecule, fromwhich the fragment is derived.

G/C modified: A G/C-modified nucleic acid may typically be a nucleicacid, preferably an artificial nucleic acid molecule as defined herein,based on a modified wild-type sequence comprising a preferably increasednumber of guanosine and/or cytosine nucleotides as compared to thewild-type sequence. Such an increased number may be generated bysubstitution of codons containing adenosine or thymidine nucleotides bycodons containing guanosine or cytosine nucleotides. If the enriched G/Ccontent occurs in a coding region of DNA or RNA, it makes use of thedegeneracy of the genetic code. Accordingly, the codon substitutionspreferably do not alter the encoded amino acid residues, but exclusivelyincrease the G/C content of the nucleic acid molecule.

Gene therapy: Gene therapy may typically be understood to mean atreatment of a patient's body or isolated elements of a patient's body,for example isolated tissues/cells, by nucleic acids encoding a peptideor protein. It may typically comprise at least one of the steps of a)administration of a nucleic acid, preferably an artificial nucleic acidmolecule as defined herein, directly to the patient—by whateveradministration route—or in vitro to isolated cells/tissues of thepatient, which results in transfection of the patient's cells either invivo/ex vivo or in vitro; b) transcription and/or translation of theintroduced nucleic acid molecule; and optionally c) re-administration ofisolated, transfected cells to the patient, if the nucleic acid has notbeen administered directly to the patient.

Genetic vaccination: Genetic vaccination may typically be understood tobe vaccination by administration of a nucleic acid molecule encoding anantigen or an immunogen or fragments thereof. The nucleic acid moleculemay be administered to a subject's body or to isolated cells of asubject. Upon transfection of certain cells of the body or upontransfection of the isolated cells, the antigen or immunogen may beexpressed by those cells and subsequently presented to the immunesystem, eliciting an adaptive, i.e. antigen-specific immune response.Accordingly, genetic vaccination typically comprises at least one of thesteps of a) administration of a nucleic acid, preferably an artificialnucleic acid molecule as defined herein, to a subject, preferably apatient, or to isolated cells of a subject, preferably a patient, whichusually results in transfection of the subject's cells, either in vivoor in vitro; b) transcription and/or translation of the introducednucleic acid molecule; and optionally c) re-administration of isolated,transfected cells to the subject, preferably the patient, if the nucleicacid has not been administered directly to the patient.

Heterologous sequence: Two sequences are typically understood to be‘heterologous’ if they are not derivable from the same gene. I.e.,although heterologous sequences may be derivable from the same organism,they naturally (in nature) do not occur in the same nucleic acidmolecule, such as in the same mRNA.

Humoral immunity/humoral immune response: Humoral immunity referstypically to antibody production and optionally to accessory processesaccompanying antibody production. A humoral immune response maytypically be characterized, e.g., by Th2 activation and cytokineproduction, germinal center formation and isotype switching, affinitymaturation and memory cell generation. Humoral immunity also typicallymay refer to the effector functions of antibodies, which includepathogen and toxin neutralization, classical complement activation, andopsonin promotion of phagocytosis and pathogen elimination.

Immunogen: In the context of the present invention, an immunogen maytypically be understood to be a compound that is able to stimulate animmune response. Preferably, an immunogen is a peptide, polypeptide, orprotein. In a particularly preferred embodiment, an immunogen in thesense of the present invention is the product of translation of aprovided nucleic acid molecule, preferably an artificial nucleic acidmolecule as defined herein. Typically, an immunogen elicits at least anadaptive immune response.

Immunostimulatory composition: In the context of the invention, animmunostimulatory composition may be typically understood to be acomposition containing at least one component, which is able to inducean immune response or from which a component which is able to induce animmune response is derivable. Such immune response may be preferably aninnate immune response or a combination of an adaptive and an innateimmune response. Preferably, an immunostimulatory composition in thecontext of the invention contains at least one artificial nucleic acidmolecule, more preferably an RNA, for example an mRNA molecule. Theimmunostimulatory component, such as the mRNA may be complexed with asuitable carrier. Thus, the immunostimulatory composition may comprisean mRNA/carrier-complex. Furthermore, the immunostimulatory compositionmay comprise an adjuvant and/or a suitable vehicle for theimmunostimulatory component, such as the mRNA.

Immune response: An immune response may typically be a specific reactionof the adaptive immune system to a particular antigen (so calledspecific or adaptive immune response) or an unspecific reaction of theinnate immune system (so called unspecific or innate immune response),or a combination thereof.

Immune system: The immune system may protect organisms from infection.If a pathogen succeeds in passing a physical barrier of an organism andenters the organism, the innate immune system provides an immediate, butnon-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained also after the pathogen has been eliminated, in the formof an immunological memory, and allows the adaptive immune system tomount faster and stronger attacks each time this pathogen isencountered. Accordingly, the immune system comprises the innate and theadaptive immune system. Each of these two parts typically contains socalled humoral and cellular components.

Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in the contextof the invention may typically be an RNA that is able to induce aninnate immune response. It usually does not have an open reading frameand thus does not provide a peptide-antigen or immunogen but elicits animmune response e.g. by binding to a specific kind of Toll-like-receptor(TLR) or other suitable receptors. However, of course also mRNAs havingan open reading frame and encoding a peptide/protein may induce aninnate immune response and, thus, may be immunostimulatory RNAs.

Innate immune system: The innate immune system, also known asnon-specific (or unspecific) immune system, typically comprises thecells and mechanisms that defend the host from infection by otherorganisms in a non-specific manner. This means that the cells of theinnate system may recognize and respond to pathogens in a generic way,but unlike the adaptive immune system, it does not confer long-lastingor protective immunity to the host. The innate immune system may be,e.g., activated by ligands of Toll-like receptors (TLRs) or otherauxiliary substances such as lipopolysaccharides, TNF-alpha, CD40ligand, or cytokines, monokines, lymphokines, interleukins orchemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF,M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of humanToll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand ofa NOD-like receptor, a ligand of a RIG-I like receptor, animmunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), aCpG-DNA, an antibacterial agent, or an anti-viral agent. Thepharmaceutical composition according to the present invention maycomprise one or more such substances. Typically, a response of theinnate immune system includes recruiting immune cells to sites ofinfection, through the production of chemical factors, includingspecialized chemical mediators, which are called cytokines; activationof the complement cascade; identification and removal of foreignsubstances present in organs, tissues, the blood and lymph, byspecialized white blood cells; activation of the adaptive immune system;and/or acting as a physical and chemical barrier to infectious agents.

Cloning site: A cloning site is typically understood to be a segment ofa nucleic acid molecule, which is suitable for insertion of a nucleicacid sequence, e.g., a nucleic acid sequence comprising an open readingframe. Insertion may be performed by any molecular biological methodknown to the one skilled in the art, e.g. by restriction and ligation. Acloning site typically comprises one or more restriction enzymerecognition sites (restriction sites). These one or more restrictionssites may be recognized by restriction enzymes, which cleave the DNA atthese sites. A cloning site, which comprises more than one restrictionsite may also be termed a multiple cloning site (MCS) or a polylinker.

Nucleic acid molecule: A nucleic acid molecule is a molecule comprising,preferably consisting of nucleic acid components. The term nucleic acidmolecule preferably refers to DNA or RNA molecules. It is preferablyused synonymously with the term “polynucleotide”. Preferably, a nucleicacid molecule is a polymer comprising or consisting of nucleotidemonomers, which are covalently linked to each other byphosphodiester-bonds of a sugar/phosphate-backbone. The term “nucleicacid molecule” also encompasses modified nucleic acid molecules, such asbase-modified, sugar-modified or backbone-modified etc. DNA or RNAmolecules.

Open reading frame: An open reading frame (ORF) in the context of theinvention may typically be a sequence of several nucleotide triplets,which may be translated into a peptide or protein. An open reading framepreferably contains a start codon, i.e. a combination of threesubsequent nucleotides coding usually for the amino acid methionine(ATG), at its 5′-end and a subsequent region, which usually exhibits alength, which is a multiple of 3 nucleotides. An ORF is preferablyterminated by a stop-codon (e.g., TAA, TAG, TGA). Typically, this is theonly stop-codon of the open reading frame. Thus, an open reading frame,in the context of the present invention, is preferably a nucleotidesequence, consisting of a number of nucleotides that may be divided bythree, which starts with a start codon (e.g. ATG) and which preferablyterminates with a stop codon (e.g., TAA, TGA, or TAG). The open readingframe may be isolated or it may be incorporated into a longer nucleicacid sequence, for example into a vector or an mRNA. An open readingframe may also be termed “protein coding region”.

Peptide: A peptide or polypeptide is typically a polymer of amino acidmonomers, linked by peptide bonds. It typically contains less than 50monomer units. Nevertheless, the term peptide is not a disclaimer formolecules having more than 50 monomer units. Long peptides are alsocalled polypeptides, typically having between 50 and 600 monomericunits.

Pharmaceutically effective amount: A pharmaceutically effective amountin the context of the invention is typically understood to be an amountthat is sufficient to induce a pharmaceutical effect, such as an immuneresponse, altering a pathological level of an expressed peptide orprotein, or substituting a lacking gene product, e.g., in case of apathological situation.

Protein: A protein typically comprises one or more peptides orpolypeptides. A protein is typically folded into 3-dimensional form,which may be required for the protein to exert its biological function.

Poly(A) sequence: A poly(A) sequence, also called poly(A) tail or3′-poly(A) tail, is usually understood to be a sequence of adeninenucleotides, e.g., of up to about 400 adenosine nucleotides, e.g. fromabout 20 to about 400, preferably from about 50 to about 400, morepreferably from about 50 to about 300, even more preferably from about50 to about 250, most preferably from about 60 to about 250 adenosinenucleotides, which is preferably added to the 3′-terminus of an mRNA. Apoly(A) sequence is typically located at the 3′-end of an mRNA. In thecontext of the present invention, a poly(A) sequence may be locatedwithin an mRNA or any other nucleic acid molecule, such as, e.g., in avector, for example, in a vector serving as template for the generationof an RNA, preferably an mRNA, e.g., by transcription of the vector. Inthe context of the present invention, the term ‘poly(A) sequence’further comprises also sequence elements, preferably artificial sequenceelements, that are part of the 3′-UTR or located at the 3′-terminus ofthe artificial nucleic acid molecule, and which preferably comprise upto 1100 adenine nucleotides, more preferably at least 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 300, 350, 400, 500, 600, 700, 800, 900, or at least 1000adenine nucleotides.

Polyadenylation: Polyadenylation is typically understood to be theaddition of a poly(A) sequence to a nucleic acid molecule, such as anRNA molecule, e.g. to a premature mRNA. Polyadenylation may be inducedby a so-called polyadenylation signal. This signal is preferably locatedwithin a stretch of nucleotides close to or at the 3′-end of a nucleicacid molecule, such as an RNA molecule, to be polyadenylated. In thecontext of the present invention, a polyadenylation signal may also becomprised by the 3′-UTR of the artificial nucleic acid molecule. Apolyadenylation signal typically comprises a hexamer consisting ofadenine and uracil/thymine nucleotides, preferably the hexamer sequenceAAUAAA. Other sequences, preferably hexamer sequences, are alsoconceivable. Polyadenylation typically occurs during processing of apre-mRNA (also called premature-mRNA). Typically, RNA maturation (frompre-mRNA to mature mRNA) comprises the step of polyadenylation. As usedin the context of the present invention, the term may relate topolyadenylation of RNA as a cellular process as well as topolyadenylation carried out by enzymatic reaction in vitro or bychemical synthesis.

Restriction site: A restriction site, also termed restriction enzymerecognition site, is a nucleotide sequence recognized by a restrictionenzyme. A restriction site is typically a short, preferably palindromic,nucleotide sequence, e.g. a sequence comprising 4 to 8 nucleotides. Arestriction site is preferably specifically recognized by a restrictionenzyme. The restriction enzyme typically cleaves a nucleotide sequencecomprising a restriction site at this site. In a double-strandednucleotide sequence, such as a double-stranded DNA sequence, therestriction enzyme typically cuts both strands of the nucleotidesequence.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic-acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually adenosine-monophosphate, uridine-monophosphate,guanosine-monophosphate and cytidine-monophosphate monomers which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificsuccession of the monomers is called the RNA sequence. Usually, RNA maybe obtainable by transcription of a DNA sequence, e.g., inside a cell.In eukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. In vivo, transcription of DNA usuallyresults in the so-called premature RNA, which has to be processed intoso-called messenger RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional-modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap, a5′-UTR, an open reading frame, a 3′-UTR and a poly(A) sequence. Asidefrom messenger RNA, several non-coding types of RNA exist, which may beinvolved in the regulation of transcription and/or translation.

Sequence of a nucleic acid molecule: The sequence of a nucleic acidmolecule is typically understood to be the particular and individualorder, i.e. the succession of its nucleotides. The sequence of a proteinor peptide is typically understood to be the order, i.e. the successionof its amino acids.

Sequence identity: Two or more sequences are identical if they exhibitthe same length and order of nucleotides or amino acids. The percentageof identity typically describes the extent, to which two sequences areidentical, i.e. it typically describes the percentage of nucleotidesthat correspond in their sequence position to identical nucleotides of areference sequence. For the determination of the degree of identity, thesequences to be compared are considered to exhibit the same length, i.e.the length of the longest sequence of the sequences to be compared. Thismeans that a first sequence consisting of 8 nucleotides is 80% identicalto a second sequence consisting of 10 nucleotides comprising the firstsequence. In other words, in the context of the present invention,identity of sequences preferably relates to the percentage ofnucleotides of a sequence, which have the same position in two or moresequences having the same length. Gaps are usually regarded asnon-identical positions, irrespective of their actual position in analignment.

Stabilized nucleic acid molecule: A stabilized nucleic acid molecule isa nucleic acid molecule, preferably a DNA or RNA molecule that ismodified such, that it is more stable to disintegration or degradation,e.g., by environmental factors or enzymatic digest, such as by an exo-or endonuclease degradation, than the nucleic acid molecule without themodification. Preferably, a stabilized nucleic acid molecule in thecontext of the present invention is stabilized in a cell, such as aprokaryotic or eukaryotic cell, preferably in a mammalian cell, such asa human cell. The stabilization effect may also be exerted outside ofcells, e.g. in a buffer solution etc., for example, in a manufacturingprocess for a pharmaceutical composition comprising the stabilizednucleic acid molecule.

Transfection: The term “transfection” refers to the introduction ofnucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, intocells, preferably into eukaryotic cells. In the context of the presentinvention, the term “transfection” encompasses any method known to theskilled person for introducing nucleic acid molecules into cells,preferably into eukaryotic cells, such as into mammalian cells. Suchmethods encompass, for example, electroporation, lipofection, e.g. basedon cationic lipids and/or liposomes, calcium phosphate precipitation,nanoparticle based transfection, virus based transfection, ortransfection based on cationic polymers, such as DEAE-dextran orpolyethylenimine etc. Preferably, the introduction is non-viral.

Vaccine: A vaccine is typically understood to be a prophylactic ortherapeutic material providing at least one antigen, preferably animmunogen. The antigen or immunogen may be derived from any materialthat is suitable for vaccination. For example, the antigen or immunogenmay be derived from a pathogen, such as from bacteria or virus particlesetc., or from a tumor or cancerous tissue. The antigen or immunogenstimulates the body's adaptive immune system to provide an adaptiveimmune response.

Vector: The term “vector” refers to a nucleic acid molecule, preferablyto an artificial nucleic acid molecule. A vector in the context of thepresent invention is suitable for incorporating or harboring a desirednucleic acid sequence, such as a nucleic acid sequence comprising anopen reading frame. Such vectors may be storage vectors, expressionvectors, cloning vectors, transfer vectors etc. A storage vector is avector, which allows the convenient storage of a nucleic acid molecule,for example, of an mRNA molecule. Thus, the vector may comprise asequence corresponding, e.g., to a desired mRNA sequence or a partthereof, such as a sequence corresponding to the open reading frame andthe 3′-UTR of an mRNA. An expression vector may be used for productionof expression products such as RNA, e.g. mRNA, or peptides, polypeptidesor proteins. For example, an expression vector may comprise sequencesneeded for transcription of a sequence stretch of the vector, such as apromoter sequence, e.g. an RNA polymerase promoter sequence. A cloningvector is typically a vector that contains a cloning site, which may beused to incorporate nucleic acid sequences into the vector. A cloningvector may be, e.g., a plasmid vector or a bacteriophage vector. Atransfer vector may be a vector, which is suitable for transferringnucleic acid molecules into cells or organisms, for example, viralvectors. A vector in the context of the present invention may be, e.g.,an RNA vector or a DNA vector. Preferably, a vector is a DNA molecule.Preferably, a vector in the sense of the present application comprises acloning site, a selection marker, such as an antibiotic resistancefactor, and a sequence suitable for multiplication of the vector, suchas an origin of replication. Preferably, a vector in the context of thepresent application is a plasmid vector.

Vehicle: A vehicle is typically understood to be a material that issuitable for storing, transporting, and/or administering a compound,such as a pharmaceutically active compound. For example, it may be aphysiologically acceptable liquid, which is suitable for storing,transporting, and/or administering a pharmaceutically active compound.

3′-untranslated region (3′-UTR): Generally, the term “3′-UTR” refers toa part of the artificial nucleic acid molecule, which is located 3′(i.e. “downstream”) of an open reading frame and which is not translatedinto protein. Typically, a 3′-UTR is the part of an mRNA, which islocated between the protein coding region (open reading frame (ORF) orcoding sequence (CDS)) and the poly(A) sequence of the mRNA. In thecontext of the invention, a 3′-UTR of the artificial nucleic acidmolecule may comprise more than one 3′-UTR elements, which may be ofdifferent origin, such as sequence elements derived from the 3′-UTR ofseveral (unrelated) naturally occuring genes. Accordingly, the term3′-UTR may also comprise elements, which are not encoded in thetemplate, from which an RNA is transcribed, but which are added aftertranscription during maturation, e.g. a poly(A) sequence. A 3′-UTR ofthe mRNA is not translated into an amino acid sequence. The 3′-UTRsequence is generally encoded by the gene, which is transcribed into therespective mRNA during the gene expression process. The genomic sequenceis first transcribed into pre-mature mRNA, which comprises optionalintrons. The pre-mature mRNA is then further processed into mature mRNAin a maturation process. This maturation process comprises the steps of5′capping, splicing the pre-mature mRNA to excize optional introns andmodifications of the 3′-end, such as polyadenylation of the 3′-end ofthe pre-mature mRNA and optional endo-/or exonuclease cleavages etc. Inthe context of the present invention, a 3′-UTR corresponds to thesequence of a mature mRNA which is located between the the stop codon ofthe protein coding region, preferably immediately 3′ to the stop codonof the protein coding region, and the poly(A) sequence of the mRNA. Theterm “corresponds to” means that the 3′-UTR sequence may be an RNAsequence, such as in the mRNA sequence used for defining the 3′-UTRsequence, or a DNA sequence, which corresponds to such RNA sequence. Inthe context of the present invention, the term “a 3′-UTR of a gene”,such as “a 3′-UTR of a ribosomal protein gene”, is the sequence, whichcorresponds to the 3′-UTR of the mature mRNA derived from this gene,i.e. the mRNA obtained by transcription of the gene and maturation ofthe pre-mature mRNA. The term “3′-UTR of a gene” encompasses the DNAsequence and the RNA sequence (both sense and antisense strand and bothmature and immature) of the 3′-UTR. As used herein, the term “3′-UTRelement” typically refers to a fragment of a 3′-UTR as defined herein.In particular, the term comprises any nucleic acid sequence element,which is located 3′ to the ORF in the artificial nucleic acid molecule,preferably the mRNA, according to the invention. Accordingly, the termcovers, for example, sequence elements derived from the 3′-UTR of aheterologous gene as well as elements such as a poly(C) sequence or ahistone stem-loop.

5′-untranslated region (5′-UTR): A 5′-UTR is typically understood to bea particular section of messenger RNA (mRNA). It is located 5′ of theopen reading frame of the mRNA. Typically, the 5′-UTR starts with thetranscriptional start site and ends one nucleotide before the startcodon of the open reading frame. The 5′-UTR may comprise elements forcontrolling gene expression, which are also called regulatory elements.Such regulatory elements may be, for example, ribosomal binding sites.The 5′-UTR may be posttranscriptionally modified, for example byaddition of a 5′-cap. In the context of the present invention, a 5′-UTRcorresponds to the sequence of a mature mRNA, which is located betweenthe 5′-cap and the start codon. Preferably, the 5′-UTR corresponds tothe sequence, which extends from a nucleotide located 3′ to the 5′-cap,preferably from the nucleotide located immediately 3′ to the 5′-cap, toa nucleotide located 5′ to the start codon of the protein coding region,preferably to the nucleotide located immediately 5′ to the start codonof the protein coding region. The nucleotide located immediately 3′ tothe 5′-CAP of a mature mRNA typically corresponds to the transcriptionalstart site. The term “corresponds to” means that the 5′-UTR sequence maybe an RNA sequence, such as in the mRNA sequence used for defining the5′-UTR sequence, or a DNA sequence, which corresponds to such RNAsequence. In the context of the present invention, the term “a 5′-UTR ofa gene” is the sequence, which corresponds to the 5′-UTR of the maturemRNA derived from this gene, i.e. the mRNA obtained by transcription ofthe gene and maturation of the pre-mature mRNA. The term “5′-UTR of agene” encompasses the DNA sequence and the RNA sequence of the 5′-UTR.

5′ Terminal Oligopyrimidine Tract (TOP): The 5′terminal oligopyrimidinetract (TOP) is typically a stretch of pyrimidine nucleotides located inthe 5′ terminal region of a nucleic acid molecule, such as the 5′terminal region of certain mRNA molecules or the 5′ terminal region of afunctional entity, e.g. the transcribed region, of certain genes. Thesequence starts with a cytidine, which usually corresponds to thetranscriptional start site, and is followed by a stretch of usuallyabout 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidinestretch and thus the 5′ TOP ends one nucleotide 5′ to the first purinenucleotide located downstream of the TOP. Messenger RNA that contains a5′terminal oligopyrimidine tract is often referred to as TOP mRNA.Accordingly, genes that provide such messenger RNAs are referred to asTOP genes. TOP sequences have, for example, been found in genes andmRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, a TOP motif is anucleic acid sequence, which corresponds to a 5′TOP as defined above.Thus, a TOP motif in the context of the present invention is preferablya stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides,preferably at least 4 pyrimidine nucleotides, preferably at least 5pyrimidine nucleotides, more preferably at least 6 nucleotides, morepreferably at least 7 nucleotides, most preferably at least 8 pyrimidinenucleotides, wherein the stretch of pyrimidine nucleotides preferablystarts at its 5′-end with a cytosine nucleotide. In TOP genes and TOPmRNAs, the TOP motif preferably starts at its 5′-end with thetranscriptional start site and ends one nucleotide 5′ to the firstpurine residue in said gene or mRNA. A TOP motif in the sense of thepresent invention is preferably located at the 5′-end of a sequence,which represents a 5′-UTR or at the 5′-end of a sequence, which codesfor a 5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidinenucleotides is called “TOP motif” in the sense of the present inventionif this stretch is located at the 5′end of a respective sequence, suchas the artificial nucleic acid molecule, the 5′-UTR element of theartificial nucleic acid molecule, or the nucleic acid sequence which isderived from the 5′-UTR of a TOP gene as described herein. In otherwords, a stretch of 3 or more pyrimidine nucleotides, which is notlocated at the 5′-end of a 5′-UTR or a 5′-UTR element but anywherewithin a 5′-UTR or a 5′-UTR element, is preferably not referred to as“TOP motif”.

TOP gene: TOP genes are typically characterised by the presence of a 5′terminal oligopyrimidine tract. Furthermore, most TOP genes arecharacterized by a growth-associated translational regulation. However,also TOP genes with a tissue specific trans-lational regulation areknown. As defined above, the 5′-UTR of a TOP gene corresponds to thesequence of a 5′-UTR of a mature mRNA derived from a TOP gene, whichpreferably extends from the nucleotide located 3′ to the 5′-cap to thenucleotide located 5′ to the start codon. A 5′-UTR of a TOP genetypically does not comprise any start codons, preferably no upstreamAUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstreamAUGs and upstream open reading frames are typically understood to beAUGs and open reading frames that occur 5′ of the start codon (AUG) ofthe open reading frame that should be translated. The 5′-UTRs of TOPgenes are generally rather short. The lengths of 5′-UTRs of TOP genesmay vary from 20 nucleotides up to 500 nucleotides, and are typicallyless than about 200 nucleotides, preferably less than about 150nucleotides, more preferably less than about 100 nucleotides. Exemplary5′-UTRs of TOP genes in the sense of the present invention are thenucleic acid sequences extending from the nucleotide at position 5 tothe nucleotide located immediately 5′ to the start codon (e.g. the ATG)in the sequences according to SEQ ID Nos. 1-1363 of the patentapplication WO2013/143700, whose disclosure is incorporated herewith byreference. In this context, a particularly preferred fragment of a5′-UTR of a TOP gene is a 5′-UTR of a TOP gene lacking the 5′TOP motif.The terms “5′-UTR of a TOP gene” or “5′-TOP UTR” preferably refer to the5′-UTR of a naturally occurring TOP gene.

In a first aspect, the present invention relates to an artificialnucleic acid molecule comprising

-   -   a) at least one open reading frame (ORF); and    -   b) a 3′-untranslated region (3′-UTR) comprising        -   b)i) at least one poly(A) sequence, wherein the at least one            poly(A) sequence comprises at least 70 adenine nucleotides,            or        -   b)ii) a polyadenylation signal.

The artificial nucleic acid molecule according to the present inventionmay be an RNA, such as an mRNA, a DNA, such as a DNA vector, or may be amodified RNA or DNA molecule. It may be provided as a double-strandedmolecule having a sense strand and an anti-sense strand, for example, asa DNA molecule having a sense strand and an anti-sense strand.

It is preferred that the artificial nucleic acid molecule according tothe invention is preferably characterized by an increased proteinexpression with respect to a reference nucleic acid molecule.

In the context of the present invention, a “reference nucleic acidmolecule” is a nucleic acid molecule, which typically comprises the sameORF as the artificial nucleic acid molecule, and which lacks a 3′-UTR orwhich comprises a reference 3′-UTR. Preferably, the reference nucleicacid molecule comprises a 3′-UTR (i.e. a “reference 3′-UTR”), whicheither does not comprise a poly(A) sequence, or which comprises apoly(A) sequence, wherein the total number of adenine nucleotidescomprised in one or more poly(A) sequence of the 3′-UTR is lower thanthe total number of adenine nucleotides comprised in the at least onepoly(A) sequence of the 3′-UTR of the artificial nucleic acid moleculeaccording to the invention, preferably lower than 70. Preferably, thereference nucleic acid molecule has—besides the different content ofadenine nucleotides in a poly(A) sequence—the same overall structure,i.e. comprises the same structural features, such as, for example, a5′-cap structure, a 5′-UTR or a 3′-UTR. More preferably, the referencenucleic acid molecule comprises or consists of—besides the differentcontent of adenine nucleotides in a poly(A) sequence—the same nucleicacid sequence as the artificial nucleic acid molecule. In the context ofthe present invention, the naturally occuring nucleic acid sequence(e.g. an mRNA) comprising the ORF of the artificial nucleic acidmolecule may also be the reference nucleic acid molecule.

In certain embodiments, a RNA molecule comprises a 5′ UTR, an ORF and 3′UTR sequence, wherein the 5′ UTR sequence or the 3′ UTR sequence isheterologous relative the ORF of the mRNA (e.g., wherein the RNA doesnot comprise the 5′ UTR sequence and/or the 3′ UTR sequence of a wildtype RNA encoding the ORF).

As used herein, the term “total number of adenine nucleotides” typicallyrefers to the sum of all adenine nucleotides, which are comprised in oneor more poly(A) sequences in the 3′-UTR of the inventive artificialnucleic acid molecule. In particular, in the case, where the 3′-UTRcomprises more than one poly(A) sequences, the term refers to the sum ofthe adenine nucleotides comprised in all poly(A) sequences comprised inthe 3′-UTR of the artificial nucleic acid molecule.

In one embodiment of the present invention, the artificial nucleic acidmolecule according to the present invention comprises a 3′-UTR, whereinthe total number of adenine nucleotides comprised in one or more poly(A)sequences of the 3′-UTR is at least 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350,400, 500, 600, 700, 800, 900, 1000 or 1100 adenine nucleotides.According to that embodiment, the 3′-UTR may comprise, for example, two,preferably separate, poly(A) sequences, wherein the sum of adeninenucleotides comprised in said two poly(A) sequences is at least 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000 or 1100.

In a further embodiment there is provided a composition comprising aplurality of RNA molecules of the embodiments in pharmaceuticallyacceptable carrier, wherein at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater of the RNA in the composition comprises apoly(A) sequence that differs in length by no more than 10 nucleotides.In a preferred embodiment at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater of the RNA in the composition comprises apoly(A) sequence of identical length. In certain embodiments, thepoly(A) sequence is positioned at the 3′ end of the RNA, with no othernucleotides positioned 3′ relative the poly(A) sequence. In still afurther embodiment, there is provided a composition comprising aplurality of RNA molecules of the embodiments in pharmaceuticallyacceptable carrier, wherein said plurality of RNA molecules compriseboth capped and uncapped RNAs. For example, in some aspects, acomposition comprises a plurality of RNA molecules wherein no more than95%, 90%, 80%, 70% or 60% of the RNAs comprise a cap and the remainingRNA molecules are uncapped.

In a preferred embodiment, the artificial nucleic acid moleculecomprises a 3′-UTR comprising at least one poly(A) sequence, wherein theat least one poly(A) sequence comprises at least 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 adeninenucleotides.

The total number of adenine nucleotides comprised in the one or morepoly(A) sequences may be up to about 1200 adenine nucleotides, e.g. fromabout 70 to about 1100, preferably from about 80 to about 800, morepreferably from about 90 to about 700, even more preferably from about100 to about 500, most preferably from about 120 to about 450 adenosinenucleotides. Preferably, the 3′-UTR of the artificial nucleic acidmolecule comprises at least 75, 80, 85, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, or at least 225, more preferably at least220, adenine nucleotides comprised in the one or more poly(A) sequences.Preferably, the 3′-UTR of the artificial nucleic acid molecule comprisesfrom 180 to 1100 adenine nucleotides comprised in the one or morepoly(A) sequences, more preferably from 200 to 1100 adenine nucleotides,even more preferably from 210 to 250 adenine nucleotides, mostpreferably from 215 to 240 adenine nucleotides. In a particularlypreferred embodiment, the 3′-UTR comprises about 220 nucleotides,preferably about 224 nucleotides. Alternatively, the 3′-UTR comprises atleast 300 adenine nucleotides comprised in the one or more poly(A)sequences, preferably at least 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, or at least 440 adenin nucleotidescomprised in the one or more poly(A) sequences. In that embodiment, the3′-UTR of the artificial nucleic acid molecule preferably comprises from300 to 650 adenine nucleotides comprised in the one or more poly(A)sequences, more preferably from 400 to 500 adenine nucleotides, evenmore preferably from 420 to 470 adenine nucleotides, most preferablyfrom 430 to 460 adenine nucleotides. In a particularly preferredembodiment, the 3′-UTR comprises a total number of about 440 adeninenucleotides, preferably about 444 adenine nucleotides, comprised in theone or more poly(A) sequences. Alternatively, the 3′-UTR comprises atleast 900 adenine nucleotides comprised in the one or more poly(A)sequences, preferably at least 900, 950 or at least 1000 adeninenucleotides comprised in the one or more poly(A) sequences.

In that embodiment, the 3′-UTR of the artificial nucleic acid moleculepreferably comprises from 900 to 1100 adenine nucleotides comprised inthe one or more poly(A) sequences. In a particularly preferredembodiment, the 3′-UTR comprises a total number of about 1064 adeninenucleotides comprised in the one or more poly(A) sequences.

In a preferred embodiment, the artificial nucleic acid moleculecomprises a 3′-UTR comprising at least one poly(A) sequence, whichcomprises at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240 or 250 adenine nucleotides, morepreferably at least 150 adenine nucleotides, even more preferably atleast 160 adenine nucleotides. Preferably, the number of adeninenucleotides comprised in the at least one poly(A) sequence is from 110to 200, from 120 to 200, from 130 to 190, from 140 to 180, or from 150to 170. Alternatively, the 3′-UTR comprises at least one poly(A)sequence, which preferably comprises at least 300 adenine nucleotides,more preferably at least 350 adenine nucleotides, even more preferablyat least 380 adenine nucleotides. Preferably, the at least one poly(A)sequence comprises from 320 to 430, from 330 to 420, from 340 to 410,from 350 to 400, from 360 to 400, or from 370 to 390. Alternatively, the3′-UTR comprises at least one poly(A) sequence, which preferablycomprises at least 900 adenine nucleotides, more preferably at least900, 950 or at least 1000 adenine nucleotides. In that embodiment, the3′-UTR of the artificial nucleic acid molecule preferably comprises atleast one poly(A) sequence, which preferably comprises from 900 to 1100or from 1000 to 1100 adenine nucleotides.

The at least one poly(A) sequence may be located at any position withinthe 3′-UTR. Thus, the at least one poly(A) sequence may be located atthe 3′ terminus of the 3′-UTR, i.e. the 3′-UTR does preferably notcontain more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotideslocated 3′ of said poly(A) sequence; more preferably the 3′-UTR does notcontain further elements located 3′ to said poly(A) sequence. In apreferred embodiment, the at least one poly(A) sequence is located atthe 3′ terminus of the artificial nucleic acid molecule, i.e. theartificial nucleic acid molecule does preferably not contain more than12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides located 3′ of saidpoly(A) sequence. Alternatively, the at least one poly(A) sequence maybe located at the 5′ terminus of the 3′-UTR, i.e. immediately 3′ of theORF of the artificial nucleic acid molecule, or located within the3′-UTR, i.e. flanked on the 5′ and on the 3′ side by other 3′-UTRelements. Preferably, the at least one poly(A) sequence is flanked onthe 3′ side by a poly(C) sequence and/or a histone stem-loop sequence.In addition or alternatively, the at least one poly(A) sequence isflanked on the 5′ side by a 3′-UTR element derived from a, preferablyhuman, albumin or globin gene, preferably albumin7 as defined herein.

In a preferred embodiment, the artificial nucleic acid moleculecomprises a 3′-UTR comprising at least one poly(A) sequence, wherein atleast one poly(A) sequence is located at the 3′ terminus of the 3′-UTRand wherein the poly(A) sequence at the 3′ terminus of the 3′-UTRcomprises at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 500, 600,700, 800, 900, or 1000 adenine nucleotides, more preferably at least 150adenine nucleotides, even more preferably at least 160 adeninenucleotides. Preferably, the number of adenine nucleotides comprised inthe poly(A) sequence located at the 3′ terminus of the 3′-UTR is from110 to 200, from 120 to 200, from 130 to 190, from 140 to 180, or from150 to 170. Alternatively, the poly(A) sequence at the 3′ terminus ofthe 3′-UTR preferably comprises at least 300 adenine nucleotides, morepreferably at least 350 adenine nucleotides, even more preferably atleast 380 adenine nucleotides. Preferably, the number of adeninenucleotides comprised in the poly(A) sequence located at the 3′ terminusof the 3′-UTR is from 320 to 430, from 330 to 450, from 340 to 410, from350 to 400, from 360 to 400, or from 370 to 390. In a preferredembodiment, the poly(A) sequence located at the 3′ terminus of the3′-UTR comprises about 160, about 380 or about 430 adenine nucleotides.Further alternatively, the poly(A) sequence at the 3′ terminus of the3′-UTR preferably comprises at least at least 900 adenine nucleotides,more preferably at least 900, 950 or at least 1000 adenine nucleotides.Preferably, the number of adenine nucleotides comprised in the poly(A)sequence located at the 3′ terminus of the 3′-UTR is from 900 to 1100 orfrom 1000 to 1100 adenine nucleotides. In a particularly preferredembodiment, the poly(A) sequence at the 3′ terminus of the 3′-UTRpreferably comprises about 1000 adenine nucleotides.

In one embodiment, the artificial nucleic acid molecule comprises a3′-UTR comprising only one poly(A) sequence and, optionally, other3′-UTR elements as defined herein. Therein, the 3′-UTR preferablyfurther comprises an optional 3′-UTR element, preferably as definedherein, a poly(C) sequence and/or a histone stem-loop sequence. In aparticularly preferred embodiment, the 3′-UTR comprises, preferably in5′ to 3′ direction, an optional 3′-UTR element, preferably as definedherein, a poly(A) sequence, a poly(C) sequence and a histone stem-loopsequence.

In another embodiment, the artificial nucleic acid molecule comprises a3′-UTR comprising at least two poly(A) sequences. Preferably, a firstpoly(A) sequence is located at the 5′ terminus of the 3′-UTR or locatedat a position within the 3′-UTR, i.e. flanked on the 5′ and on the 3′side by other UTR-elements, while a second poly(A) sequence is locatedat a position within the 3′-UTR or at the 3′ terminus of the 3′-UTR.Preferably, the first poly(A) sequence is flanked on the 3′ side by apoly(C) sequence and/or a histone stem-loop sequence. In addition oralternatively, the first poly(A) sequence is flanked on the 5′ side by a3′-UTR element derived from a, preferably human, albumin or globin gene,preferably albumin7 as defined herein.

In a preferred embodiment, the artificial nucleic acid moleculecomprises at least two poly(A) sequences, which are separated from eachother by a nucleotide sequence comprising or consisting of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145or 150 nucleotides, wherein the nucleotide sequence does preferably notcomprise more than 10, 9, 8, 7, 6, 5, 4, 3 or 2 consecutive adeninenucleotides. Preferably, the nucleotide sequence, which separates thefirst and the second poly(A) sequence comprises from 1 to about 200nucleotides, preferably from 10 to 90, from 20 to 85, from 30 to 80,from 40 to 80, from 50 to 75 or from 55 to 85 nucleotides, morepreferably from 55 to 80 nucleotides, wherein the nucleotide sequencedoes preferably not comprise more than 10, 9, 8, 7, 6, 5, 4, 3 or 2consecutive adenine nucleotides.

Preferably, the artificial nucleic acid molecule comprises a 3′-UTRcomprising at least two poly(A) sequences, wherein the first and/or thesecond poly(A) sequence preferably comprises at least 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 adeninenucleotides, more preferably at least 150 adenine nucleotides, even morepreferably at least 160 adenine nucleotides. In a preferred embodiment,the first poly(A) sequence comprises at least 20, 30, 40, 50, 60, 70, 80or 90 adenine nucleotides. The first poly(A) sequence may furthercomprise from 20 to 90, from 25 to 85, from 35 to 80 or from 45 to 75,preferably from 60 to 70, adenine nucleotides. In a further preferredembodiment, the first poly(A) sequence comprises about 60 adeninenucleotides. In a particularly preferred embodiment, the first poly(A)sequence comprises or consists of about 64 adenine nucleotides. Thesecond poly(A) sequence preferably comprises at least 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 adeninenucleotides, more preferably at least 150 adenine nucleotides, even morepreferably at least 160 adenine nucleotides. In a particularly preferredembodiment, the second poly(A) sequence comprises about 160 adeninenucleotides. Alternatively, the second poly(A) sequence preferablycomprises at least 300 adenine nucleotides, more preferably at least 350adenine nucleotides, even more preferably at least 380 adeninenucleotides or at least 430 adenine nucleotides. In a preferredembodiment, the second poly(A) sequence comprises about 380 or about 430adenine residues. Preferably, the number of adenine nucleotidescomprised in the second poly(A) sequence is preferably from 110 to 200,from 120 to 200, from 130 to 190, from 140 to 180, or from 150 to 170.Alternatively, the number of adenine nucleotides comprised in the secondpoly(A) sequence is preferably from 320 to 450, from 330 to 420, from340 to 410, from 350 to 400, from 360 to 400, or from 370 to 390.Further alternatively, the second poly(A) sequence preferably comprisesat least 900 adenine nucleotides, more preferably at least 900, 950 orat least 1000 adenine nucleotides. Preferably, the number of adeninenucleotides comprised in the second poly(A) sequence is from 900 to 1100or from 1000 to 1100 adenine nucleotides. In a particularly preferredembodiment, the second poly(A) sequence preferably comprises about 1000adenine nucleotides.

In a preferred embodiment, the artificial nucleic acid moleculecomprises a 3′-UTR comprising at least two poly(A) sequences, whereinthe first poly(A) sequence is located at the 5′ terminus of the 3′-UTRor located at a position within the 3′-UTR, i.e. flanked on the 5′ andon the 3′ side by other UTR-elements, and comprises at least 20, 30, 40,50, 60, 70, 80 or 90 adenine nucleotides, preferably from 20 to 90, from25 to 85, from 35 to 80 or from 45 to 75, preferably from 60 to 70, morepreferably about 64, adenine nucleotides. In that embodiment, the secondpoly(A) sequence preferably comprises at least 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,300, 350, 400, 500, 600, 700, 800, 900, or 1000 adenine nucleotides,more preferably at least 150 adenine nucleotides, even more preferablyat least 160 adenine nucleotides, preferably from 110 to 200, from 120to 200, from 130 to 190, from 140 to 180, or from 150 to 170 adeninenucleotides. Therein, the second poly(A) sequence is preferably located3′ of the first poly(A) sequence, more preferably at the 3′-terminus ofthe 3′-UTR as defined herein, or even more preferably at the 3′-terminusof the inventive artificial nucleic acid molecule as defined herein,wherein the first and the second poly(A) sequences are preferablyseparated, more preferably separated as defined herein.

In a further preferred embodiment, the artificial nucleic acid moleculecomprises a 3′-UTR comprising at least two poly(A) sequences, whereinthe first poly(A) sequence is located at the 5′ terminus of the 3′-UTRor located at a position within the 3′-UTR, i.e. flanked on the 5′ andon the 3′ side by other UTR-elements, and comprises at least 20, 30, 40,50, 60, 70, 80 or 90 adenine nucleotides, preferably from 20 to 90, from25 to 85, from 35 to 80 or from 45 to 75, preferably from 60 to 70, morepreferably about 64, adenine nucleotides. In that embodiment, the secondpoly(A) sequence preferably comprises at least 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,300, 350, 400, 500, 600, 700, 800, 900, or 1000, more preferably 300adenine nucleotides, further preferably at least 350 adeninenucleotides, even more preferably at least 380 adenine nucleotides,preferably from 320 to 430, from 330 to 420, from 340 to 410, from 350to 400, from 360 to 400, or from 370 to 390 adenine nucleotides.Therein, the second poly(A) sequence is preferably located 3′ of thefirst poly(A) sequence, more preferably at the 3′-end of the 3′-UTR ofthe inventive artificial nucleic acid molecule, wherein the first andthe second poly(A) sequences are preferably separated, more preferablyseparated as defined herein.

In a further preferred embodiment, the artificial nucleic acid moleculecomprises a 3′-UTR comprising at least two poly(A) sequences, whereinthe first poly(A) sequence is located at the 5′ terminus of the 3′-UTRor located at a position within the 3′-UTR, i.e. flanked on the 5′ andon the 3′ side by other UTR-elements, and comprises at least 20, 30, 40,50, 60, 70, 80 or 90 adenine nucleotides, preferably from 20 to 90, from25 to 85, from 35 to 80 or from 45 to 75, preferably from 60 to 70, morepreferably about 64, adenine nucleotides. In that embodiment, the secondpoly(A) sequence preferably comprises at least 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,300, 350, 400, 500, 600, 700, 800, 900, or 1000, more preferably atleast 900 adenine nucleotides, more preferably at least 900, at least950 or at least 1000 adenine nucleotides. Preferably, the number ofadenine nucleotides comprised in the second poly(A) sequence is from 900to 1100 or from 1000 to 1100 adenine nucleotides. In a particularlypreferred embodiment, the second poly(A) sequence preferably comprisesabout 1000 adenine nucleotides. Therein, the second poly(A) sequence ispreferably located 3′ of the first poly(A) sequence, more preferably atthe 3′-end of the 3′-UTR of the inventive artificial nucleic acidmolecule, wherein the first and the second poly(A) sequences arepreferably separated, more preferably separated as defined herein.

An artificial nucleic acid molecule according to the invention, such asa DNA molecule comprising an ORF followed by a 3′-UTR, may contain atleast one stretch of thymidine nucleotides, which corresponds to the atleast one poly(A) sequence as defined herein and which can betranscribed into a poly(A) sequence as defined herein in the resultingmRNA.

For example, the artificial nucleic acid molecule according to thepresent invention may comprise a nucleic acid sequence corresponding tothe DNA sequence

(SEQ ID No. 1) GTCCACCTGTCCCTCCTGGGCTGCTGGATTGTCTCGTTTTCCTGCCAAATAAACAGGATCAGCGCTTTACAGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

Transcription of such sequences may result in artificial nucleic acidmolecules comprising the sequence

(SEQ ID No. 2) GUCCACCUGUCCCUCCUGGGCUGCUGGAUUGUCUCGUUUUCCUGCCAAAUAAACAGGAUCAGCGCUUUACAGAUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

Such artificial RNA molecules, i.e. artificial nucleic acid moleculescomprising a sequence according to SEQ ID No. 2 may also be obtainablein vitro by common methods of chemical-synthesis without beingnecessarily transcribed from a DNA-progenitor.

Alternatively or in addition, multiple adenine nucleotides may be addedto an artificial nucleic acid molecule according to the invention by anyother technique known in the art, for example via chemical synthesis orvia an adenylation reaction. Preferably, adenine nucleotides are addedto an inventive artificial nucleic acid molecule as described herein byan enzymatic adenylation reaction. For instance, an inventive RNAmolecule may be enzymatically polyadenylated by incubation with asuitable enzyme, such as E. coli poly(A) polymerase.

In one embodiment, the artificial nucleic acid molecule according to theinvention comprises an ORF and a 3′-UTR, wherein the 3′-UTR comprises apolyadenylation signal. In the context of the present invention, thepolyadenylation signal is located within the 3′-UTR, at the 3′-terminusof the 3′-UTR or downstream of the 3′ terminus of the 3′-UTR element.Preferably, the polyadenylation signal as used herein is comprised bythe 3′-UTR of the artificial nucleic acid molecule. Even morepreferably, the polyadenylation signal is located at the 3′ terminus ofthe 3′-UTR, i.e. the 3′-UTR does preferably not contain more than 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides located 3′ of saidpolyadenylation signal; more preferably the 3′-UTR does not containfurther elements located 3′ to said polyadenylation signal. In apreferred embodiment, the polyadenylation signal is located at the 3′terminus of the artificial nucleic acid molecule, i.e. the artificialnucleic acid molecule does preferably not contain more than 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides located 3′ of said poly(A)sequence.

In a preferred embodiment, the artificial nucleic acid moleculeaccording to the invention comprises a 3′-UTR, which comprises at leastone poly(A) sequence, preferably as defined herein, and apolyadenylation signal. Therein, the polyadenylation signal ispreferably located 3′ of the at least one poly(A) sequence comprised inthe 3′-UTR, more preferably located 3′ of the most 3′ poly(A) sequencein the 3′-UTR. More preferably, the polyadenylation signal is located 3′of the at least one poly(A) signal, more preferably 3′ of the most 3′poly(A) sequence, and is separated from the said poly(A) signal by anucleotide sequence comprising or consisting of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150nucleotides, wherein the nucleotide sequence does preferably notcomprise more than 10, 9, 8, 7, 6, 5, 4, 3 or 2 consecutive adeninenucleotides. Preferably, the nucleotide sequence, which separates the atleast one poly(A) sequence and the polyadenylation signal comprises from1 to about 200 nucleotides, preferably from 10 to 90, from 20 to 85,from 30 to 80, from 40 to 80, from 50 to 75 or from 55 to 85nucleotides, more preferably from 55 to 80 nucleotides, wherein thenucleotide sequence does preferably not comprise more than 10, 9, 8, 7,6, 5, 4, 3 or 2 consecutive adenine nucleotides.

In a preferred embodiment, the artificial nucleic acid moleculeaccording to the invention comprises a 3′-UTR, which comprises at leasttwo poly(A) sequences, preferably as defined herein, and apolyadenylation signal, wherein the polyadenylation is located 3′ of theat least two poly(A) sequences, and wherein preferably each of the atleast two poly(A) sequences and the polyadenylation signal is separatedfrom another poly(A) sequence or from the polyadenylation signal,respectively, wherein the separating nucleotide sequences are as definedabove. Therein, the polyadenylation signal is preferably located at the3′ terminus of the 3′-UTR as defined herein, more preferably at the 3′terminus of the artificial nucleic acid molecule as defined herein.

In one embodiment, the artificial nucleic acid molecule is a DNAmolecule comprising a 3′-UTR comprising a polyadenylation signal,preferably as defined herein, wherein the DNA molecule furtheroptionally comprises at least one poly(A) sequence, preferably asdefined herein. Alternatively, the artificial nucleic acid molecule isan RNA molecule comprising a 3′-UTR comprising a polyadenylation signal,preferably as defined herein, wherin the RNA molecule optionally furthercomprises at least one poly(A) sequence, preferably as defined herein.

The polyadenylation signal preferably comprises the consensus sequenceNN(U/T)ANA, with N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA. Suchconsensus sequence may be recognised by most animal and bacterialcell-systems, for example by the polyadenylation-factors, such ascleavage/polyadenylation specificity factor (CPSF) cooperating withCstF, PAP, PAB2, CFI and/or CFII. Preferably, the polyadenylationsignal, preferably the consensus sequence NNUANA, is located less thanabout 50 nucleotides, more preferably less than about 30 bases, mostpreferably less than about 25 bases, for example 21 bases, downstream ofthe 3′-end of the optional 3′-UTR element as defined herein. Furtherpreferably, the polyadenylation signal, preferably the consensussequence defined above, is located as described above.

Transcription of an artificial nucleic acid molecule according to thepresent invention, e.g. of an artificial DNA molecule comprising apolyadenylation signal downstream (i.e. in the 3′ direction) of the3′-UTR will result in a premature-RNA containing the polyadenylationsignal downstream of its 3′-UTR.

Using an appropriate transcription system will then lead to theattachment of a poly(A) sequence to the premature-RNA. For example, theinventive artificial nucleic acid molecule may be a DNA moleculecomprising a 3′-UTR as described herein comprising a polyadenylationsignal, which may result in polyadenylation of an RNA upon transcriptionof this DNA molecule. Accordingly, a resulting RNA may comprise a3′-UTR, which comprises at least one poly(A) sequence, and wherein the3′-UTR is followed by an additional poly(A) sequence.

Potential transcription systems are in vitro transcription systems orcellular transcription systems etc. Accordingly, transcription of anartificial nucleic acid molecule according to the invention, e.g.transcription of an artificial nucleic acid molecule comprising an openreading frame, a 3′-UTR as defined herein comprising apolyadenylation-signal, may result in an mRNA molecule comprising anopen reading frame, a 3′-UTR as defined herein comprising an additionalpoly(A) sequence.

The inventors have surprisingly found that the 3′-UTR comprising atleast one poly(A) sequence as defined herein or a polyadenylation signalas defined herein results in an increased expression of the proteinencoded by the ORF of the artificial nucleic acid molecule.

“Increased protein expression” or “enhanced protein expression” in thecontext of the present invention preferably means an increased/enhancedprotein expression at one time point after initiation of expression oran increased/enhanced total amount of expressed protein compared to theexpression induced by a reference nucleic acid molecule. Thus, theprotein level observed at a certain time point after initiation ofexpression, e.g. after transfection, of the artificial nucleic acidmolecule according to the present invention or after administration,e.g. by injection, of the artificial nucleic acid molecule to a tissue,e.g. after transfection or administration of an mRNA according to thepresent invention, for example, 6, 12, 24, 48 or 72 hours posttransfection or administration, respectively, is preferably higher thanthe protein level observed at the same time point after initiation ofexpression, e.g. after transfection or administration, of a referencenucleic acid molecule, such as a reference mRNA comprising a reference3′-UTR or lacking a 3′-UTR. In a preferred embodiment, the maximumamount of protein (as determined e.g. by protein activity or mass)expressed from the artificial nucleic acid molecule is increased withrespect to the protein amount expressed from a reference nucleic acidcomprising a reference 3′-UTR or lacking a 3′-UTR. Peak expressionlevels are preferably reached within 48 hours, more preferably within 24hours and even more preferably within 12 hours after, for instance,transfection or administration to a tissue.

Preferably, the total protein production from an artificial nucleic acidmolecule according to the invention is increased or enhanced withrespect to a reference nucleic acid. In particular, protein productionis preferably increased or enhanced over the time span, in which proteinis produced from an artificial nucleic acid molecule, preferably in atarget tissue or in a mammalian expression system, such as in mammaliancells, e.g. in HeLa or HDF cells in comparison to a reference nucleicacid molecule lacking a 3′-UTR or comprising a reference 3′-UTR.According to a preferred embodiment, the cumulative amount of proteinexpressed over time is increased when using the artificial nucleic acidmolecule according to the invention.

The artificial nucleic acid molecule according to the invention ispreferably characterized by increased expression of the encoded proteinin comparison to a respective nucleic acid molecule lacking the at leastone 3′-UTR element or comprising a reference 3′-UTR (“reference nucleicacid”) comprising a nucleic acid sequence which is derived from the3′-UTR of a ribosomal protein gene or from a variant of the 3′-UTR of aribosomal protein gene In order to assess the in vivo protein productionby the inventive artificial nucleic acid molecule, the expression of theencoded protein is determined following injection/transfection of theinventive artificial nucleic acid molecule into target cells/tissue andcompared to the protein expression induced by the reference nucleicacid. Quantitative methods for determining protein expression are knownin the art (e.g. Western-Blot, FACS, ELISA, mass spectometry).Particularly useful in this context is the determination of theexpression of reporter proteins like luciferase, Green fluorescentprotein (GFP), or secreted alkaline phosphatase (SEAP). Thus, anartificial nucleic acid according to the invention or a referencenucleic acid is introduced into the target tissue or cell, e.g. viatransfection or injection. Several hours or several days (e.g. 6, 12,24, 48 or 72 hours) post initiation of expression or post introductionof the nucleic acid molecule, a target cell sample is collected andmeasured via FACS and/or lysed. Afterwards the lysates can be used todetect the expressed protein (and thus determine the efficiency ofprotein expression) using several methods, e.g. Western-Blot, FACS,ELISA, mass spectrometry or by fluorescence or luminescence measurement.

Therefore, if the protein expression from an artificial nucleic acidmolecule according to the invention is compared to the proteinexpression from a reference nucleic acid molecule at a specific timepoint (e.g. 6, 12, 24, 48 or 72 hours post initiation of expression orpost introduction of the nucleic acid molecule), both nucleic acidmolecules are introduced separately into target tissue/cells, a samplefrom the tissue/cells is collected after a specific time point, proteinlysates are prepared according to the particular protocol adjusted tothe particular detection method (e.g. Western Blot, ELISA, etc. as knownin the art) and the protein is detected by the chosen detection method.As an alternative to the measurement of expressed protein amounts incell lysates—or, in addition to the measurement of protein amounts incell lysates prior to lysis of the collected cells or using an aliquotin parallel—protein amounts may also be determined by using FACSanalysis.

If the total amount of protein for a specific time period is to bemeasured, tissue or cells can be collected after several time pointsafter introduction of the artificial nucleic acid molecule (e.g. 6, 12,24, 48 and 72 hours post initiation of expression or post introductionof the nucleic acid molecule; usually from different test animals), andthe protein amount per time point can be determined as explained above.In order to calculate the cumulative protein amount, a mathematicalmethod of determining the total amount of protein can be used, e.g. thearea under the curve (AUC) can be determined according to the followingformula:

$\begin{matrix}{{AUC} = {\int\limits_{a}^{b}{{f(x)}{d(x)}}}} & \;\end{matrix}$

In order to calculate the area under the curve for total amount ofprotein, the integral of the equation of the expression curve from eachend point (a and b) is calculated.

Thus, “total protein production” preferably refers to the area under thecurve (AUC) representing protein production over time.

Preferably, the nucleic acid molecule according to the inventioncomprises a 3′-UTR comprising at least one poly(A) sequence as definedherein, which optionally further comprises at least one further 3′-UTRelement, which is distinct from a poly(A) sequence. Therein, the atleast one further 3′-UTR element preferably increases protein expressionfrom said artificial nucleic acid molecule.

In this context, the term “3′-UTR element” typically refers to anoptional element of the 3′-UTR of the artificial nucleic acid moleculeaccording to the invention, wherein the 3′-UTR element is a nucleic acidsequence that is distinct from a poly(A) sequence, i.e. is not a poly(A)sequence. In particular, the term refers to a nucleic acid sequence,which comprises or consists of a nucleic acid sequence that is derivedfrom a 3′-UTR or from a variant of a 3′-UTR. A “3′-UTR element”preferably refers to a nucleic acid sequence, which represents a3′-UTR—or a part thereof—of an artificial nucleic acid sequence, such asan artificial mRNA, or which codes for a 3′-UTR of an artificial nucleicacid molecule. Accordingly, in the sense of the present invention,preferably, a 3′-UTR element may be the 3′-UTR of an mRNA, preferably ofan artificial mRNA, or it may be the transcription template for a 3′-UTRof an mRNA. Thus, a 3′-UTR element preferably is a nucleic acidsequence, which corresponds to the 3′-UTR of an mRNA, preferably to the3′-UTR of an artificial mRNA, such as an mRNA obtained by transcriptionof a genetically engineered vector construct. Preferably, a 3′-UTRelement in the sense of the present invention functions as a 3′-UTR orcodes for a nucleotide sequence that fulfils the function of a 3′-UTR.

Preferably, the at least one open reading frame and the 3′-UTR areheterologous. The term “heterologous” in this context means that theopen reading frame and the 3′-UTR or the 3′-UTR element are notoccurring naturally (in nature) in this combination. Preferably, the3′-UTR or the 3′-UTR element is derived from a different gene than theopen reading frame. For example, the ORF may be derived from a differentgene than the 3′-UTR, e.g. encoding a different protein or the sameprotein but of a different species etc. Preferably, the open readingframe does not code for a ribosomal protein. In specific embodiments itis preferred that the open reading frame does not code for a reporterprotein, e.g., selected from the group consisting of globin proteins(particularly beta-globin), luciferase protein, GFP proteins or variantsthereof, for example, variants exhibiting at least 70% sequence identityto a globin protein, a luciferase protein, or a GFP protein.

Preferably, the at least one 3′-UTR is functionally linked to the ORF.This means preferably that the 3′-UTR is associated with the ORF suchthat it may exert a function, such as an increasing, enhancing orstabilizing function on the expression of the encoded peptide or proteinor a stabilizing function on the artificial nucleic acid molecule.Preferably, the ORF and the 3′-UTR are associated in 5′43′ direction.Thus, preferably, the artificial nucleic acid molecule comprises thestructure 5′-ORF-(optional)-linker-3′-UTR-3′, wherein the linker may bepresent or absent. For example, the linker may be one or morenucleotides, such as a stretch of 1 to 50 or 1 to 20 nucleotides, e.g.,comprising or consisting of one or more restriction enzyme recognitionsites (restriction sites).

In one embodiment, the 3′-UTR of the inventive artificial nucleic acidmolecule optionally comprises a 3′-UTR element, which may be derivablefrom a gene that relates to an mRNA with an enhanced half-life (thatprovides a stable mRNA), for example, a 3′UTR element as defined anddescribed below.

In a particularly preferred embodiment, the optional 3′-UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 3′-UTR of a gene selected from the group consisting of an albumingene, an α-globin gene, a β-globin gene, a ribosomal protein gene, atyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alphagene, such as a collagen alpha 1(l) gene, or from a variant of a 3′-UTRof a gene selected from the group consisting of an albumin gene, anα-globin gene, a β-globin gene, a ribosomal protein gene, a tyrosinehydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, suchas a collagen alpha 1(l) gene according to SEQ ID No. 1369-1390 of thepatent application WO2013/143700, whose disclosure is incorporatedherein by reference. In a particularly preferred embodiment, the 3′-UTRelement comprises or consists of a nucleic acid sequence, which isderived from a 3′UTR of an albumin gene, preferably a vertebrate albumingene, more preferably a mammalian albumin gene, most preferably a humanalbumin gene according to SEQ ID No. 3

  Human albumin 3′-UTR SEQ ID No. 3:CATCACATTT AAAAGCATCT CAGCCTACCA TGAGAATAAGAGAAAGAAAA TGAAGATCAA AAGCTTATTC ATCTGTTTTTCTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAACATAAATTTCT TTAATCATTT TGCCTCTTTT CTCTGTGCTT CAATTAATAA AAAATGGAAA GAATCT(corresponding to SEQ ID No: 1369 ofthe patent application WO2013/143700).

In this context, it is particularly preferred that the 3′-UTR of theinventive artificial nucleic acid molecule optionally comprises a 3′-UTRelement comprising a corresponding RNA sequence derived from the nucleicacids according to SEQ ID No. 1369-1390 of the patent applicationWO2013/143700 or a fragment, homolog or variant thereof.

Most preferably, the optional 3′-UTR element comprises the nucleic acidsequence derived from a fragment of the human albumin gene according toSEQ ID No. 4:

albumin7 3′UTR CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCT(SEQ ID No. 4 corresponding to SEQ ID No:1376 of the patent application WO2013/143700)

In this context, it is particularly preferred that the optional 3′-UTRelement of the inventive artificial nucleic acid molecule comprises orconsists of a corresponding RNA sequence of the nucleic acid sequenceaccording to SEQ ID No. 4.

In another particularly preferred embodiment, the 3′UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 3′UTR of an α-globin gene, preferably a vertebrate α- or β-globingene, more preferably a mammalian α- or β-globin gene, most preferably ahuman α- or β-globin gene according to SEQ ID No. 5-7:

3′-UTR of Homo sapiens hemoglobin, alpha 1 (HBA1) GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG AGTGGGCGGC (SEQ ID No: 5 corresponding to SEQ ID No.1370 of the patent application WO2013/143700) 3′-UTR of Homo sapiens hemoglobin, alpha 2 (HBA2) GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAAGTCTGA GTGGGCAG (SEQ ID No: 6 corresponding to SEQ ID No.1371 of the patent application WO2013/143700) 3′-UTR of Homo sapiens hemoglobin, beta (HBB) GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC(SEQ ID No: 7 corresponding to SEQ ID No.1372 of the patent application WO2013/143700). 

For example, the optional 3′UTR element may comprise or consist of thecenter, α-complex-binding portion of the 3′UTR of an α-globin gene, suchas of a human α-globin gene, preferably according to SEQ ID No. 8:

Center, α-complex-binding portion of the 3′UTR of an α-globin gene (alsonamed herein as “muag”)

  GCCCGATGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCG(SEQ ID NO. 8 corresponding to SEQ ID No. 1393 of the patent applicationWO2013/143700). 

In this context, it is particularly preferred that the 3′-UTR element ofthe inventive mRNA comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID No. 8 or a homolog, afragment or variant thereof.

In a preferred embodiment, the (optional) at least one 3′-UTR elementcomprises a nucleic acid sequence, which is derived from the 3′-UTR of aeukaryotic ribosomal protein gene, preferably from the 3′-UTR of avertebrate ribosomal protein gene, more preferably from the 3′-UTR of amammalian ribosomal protein gene, even more preferably from the 3′-UTRof a primate ribosomal protein gene, in particular of a human ribosomalprotein gene.

In a preferred embodiment, the optional 3′-UTR element comprises orcorresponds to a nucleic acid sequence, which is derived from the 3′-UTRsequence of a transcript selected from the group consisting ofNM_000661.4, NM_001024921.2, NM_000967.3, NM_001033853.1, NM_000968.3,NM_000969.3, NM_001024662.1, NM_000970.3, NM_000971.3, NM_000972.2,NM_000975.3, NM_001199802.1, NM_000976.3, NM_000977.3, NM_033251.2,NM_001243130.1, NM_001243131, NM_000978.3, NM_000979.3, NM_001270490.1,NM_000980.3, NM_000981.3, NM_000982.3, NM_000983.3, NM_000984.5,NM_000985.4, NM_001035006.2, NM_001199340.1, NM_001199341.1,NM_001199342.1, NM_001199343.1, NM_001199344.1, NM_001199345.1,NM_000986.3, NM_000987.3, NM_000988.3, NM_000989.3, NM_000990.4,NM_001136134.1, NM_000991.4, NM_001136135.1, NM_001136136.1,NM_001136137.1, NM_000992.2, NM_000993.4, NM_001098577.2,NM_001099693.1, NM_000994.3, NM_001007073.1, NM_001007074.1,NM_000996.2, NM_000997.4, NM_000998.4, NM_000999.3, NM_001035258.1,NM_001000.3, NM_001002.3, NM_053275.3, NM_001003.2, NM_213725.1,NM_001004.3, NM_001005.4, NM_001256802.1, NM_001260506.1,NM_001260507.1, NM_001006.4, NM_001267699.1, NM_001007.4, NM_001008.3,NM_001009.3, NM_001010.2, NM_001011.3, NM_001012.1, NM_001013.3,NM_001203245.2, NM_001014.4, NM_001204091.1, NM_001015.4, NM_001016.3,NM_001017.2, NM_001018.3, NM_001030009.1, NM_001019.4, NM_001020.4,NM_001022.3, NM_001146227.1, NM_001023.3, NM_001024.3, NM_001025.4,NM_001028.2, NM_001029.3, NM_001030.4, NM_002954, NM_001135592.2,NM_001177413.1, NM_001031.4, NM_001032.4, NM_001030001.2, NM_002948.3,NM_001253379.1, NM_001253380.1, NM_001253382.1, NM_001253383.1,NM_001253384.1, NM_002952.3, NM_001034996.2, NM_001025071.1,NM_001025070.1, NM_005617.3, NM_006013.3, NM_001256577.1,NM_001256580.1, NM_007104.4, NM_007209.3, NM_012423.3, NM_001270491.1,NM_033643.2, NM_015414.3, NM_021029.5, NM_001199972.1, NM_021104.1,NM_022551.2, NM_033022.3, NM_001142284.1, NM_001026.4, NM_001142285.1,NM_001142283.1, NM_001142282.1, NM_000973.3, NM_033301.1, NM_000995.3,NM_033625.2, NM_001021.3, NM_002295.4, NM_001012321.1, NM_001033930.1,NM_003333.3, NM_001997.4, NM_001099645.1, NM_001021.3, NM_052969.1,NM_080746.2, NM_001001.4, NM_005061.2, NM_015920.3, NM_016093.2,NM_198486.2, NG_011172.1, NG_011253.1, NG_000952.4, NR_002309.1,NG_010827.2, NG_009952.2, NG_009517.1

In a preferred embodiment, the 3′-UTR element of the artificial nucleicacid molecule according to the present invention is derived from the3′-UTR region of a gene encoding a ribosomal protein, preferably fromthe 3′-UTR region of ribosomal protein L9 (RPL9), ribosomal protein L3(RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5),ribosomal protein L6 (RPL6), ribosomal protein L7 (RPL7), ribosomalprotein L7a (RPL7A), ribosomal protein L11 (RPL11), ribosomal proteinL12 (RPL12), ribosomal protein L13 (RPL13), ribosomal protein L23(RPL23), ribosomal protein L18 (RPL18), ribosomal protein L18a (RPL18A),ribosomal protein L19 (RPL19), ribosomal protein L21 (RPL21), ribosomalprotein L22 (RPL22), ribosomal protein L23a (RPL23A), ribosomal proteinL17 (RPL17), ribosomal protein L24 (RPL24), ribosomal protein L26(RPL26), ribosomal protein L27 (RPL27), ribosomal protein L30 (RPL30),ribosomal protein L27a (RPL27A), ribosomal protein L28 (RPL28),ribosomal protein L29 (RPL29), ribosomal protein L31 (RPL31), ribosomalprotein L32 (RPL32), ribosomal protein L35a (RPL35A), ribosomal proteinL37 (RPL37), ribosomal protein L37a (RPL37A), ribosomal protein L38(RPL38), ribosomal protein L39 (RPL39), ribosomal protein, large, P0(RPLP0), ribosomal protein, large, P1 (RPLP1), ribosomal protein, large,P2 (RPLP2), ribosomal protein S3 (RPS3), ribosomal protein S3A (RPS3A),ribosomal protein S4, X-linked (RPS4X), ribosomal protein S4, Y-linked 1(RPS4Y1), ribosomal protein S5 (RPS5), ribosomal protein S6 (RPS6),ribosomal protein S7 (RPS7), ribosomal protein S8 (RPS8), ribosomalprotein S9 (RPS9), ribosomal protein S10 (RPS10), ribosomal protein S11(RPS11), ribosomal protein S12 (RPS12), ribosomal protein S13 (RPS13),ribosomal protein S15 (RPS15), ribosomal protein S15a (RPS15A),ribosomal protein S16 (RPS16), ribosomal protein S19 (RPS19), ribosomalprotein S20 (RPS20), ribosomal protein S21 (RPS21), ribosomal proteinS23 (RPS23), ribosomal protein S25 (RPS25), ribosomal protein S26(RPS26), ribosomal protein S27 (RPS27), ribosomal protein S27a (RPS27a),ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29), ribosomalprotein L15 (RPL15), ribosomal protein S2 (RPS2), ribosomal protein L14(RPL14), ribosomal protein S14 (RPS14), ribosomal protein L10 (RPL10),ribosomal protein L10a (RPL10A), ribosomal protein L35 (RPL35),ribosomal protein L13a (RPL13A), ribosomal protein L36 (RPL36),ribosomal protein L36a (RPL36A), ribosomal protein L41 (RPL41),ribosomal protein S18 (RPS18), ribosomal protein S24 (RPS24), ribosomalprotein L8 (RPL8), ribosomal protein L34 (RPL34), ribosomal protein S17(RPS17), ribosomal protein SA (RPSA) or ribosomal protein S17 (RPS17).In an alternative embodiment, the 3′-UTR element may be derived from agene encoding a ribosomal protein or from a gene selected from ubiquitinA-52 residue ribosomal protein fusion product 1 (UBA52),Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitouslyexpressed (FAU), ribosomal protein L22-like 1 (RPL22L1), ribosomalprotein L39-like (RPL39L), ribosomal protein L10-like (RPL10L),ribosomal protein L36a-like (RPL36AL), ribosomal protein L3-like(RPL3L), ribosomal protein S27-like (RPS27L), ribosomal protein L26-like1 (RPL26L1), ribosomal protein L7-like 1 (RPL7L1), ribosomal proteinL13a pseudogene (RPL13AP), ribosomal protein L37a pseudogene 8(RPL37AP8), ribosomal protein S10 pseudogene 5 (RPS10P5), ribosomalprotein S26 pseudogene 11 (RPS26P11), ribosomal protein L39 pseudogene 5(RPL39P5), ribosomal protein, large, P0 pseudogene 6 (RPLP0P6) andribosomal protein L36 pseudogene 14 (RPL36P14).

Preferably, the at least one 3′-UTR element of the artificial nucleicacid molecule according to the present invention comprises or consistsof a nucleic acid sequence which has an identity of at least about 1, 2,3, 4, 5, 10, 15, 20, 30 or 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99%, most preferably of 100% to the nucleic acid sequence ofa 3′-UTR of a ribosomal protein gene, such as to the nucleic acidsequences according to SEQ ID NOs:10 to 115 as defined in internationalpatent application PCT/EP2013/003946, or the corresponding RNA sequence.

In a preferred embodiment, the (optional) at least one 3′-UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99%, most preferably of 100% to the 3′-UTR sequence of ribosomal proteinSmall 9 (RPS9). Most preferably, the (optional) at least one 3′-UTRelement comprises or consists of a nucleic acid sequence which has anidentity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99%, most preferably of 100% to SEQ ID NO: 9 or SEQ ID NO:10

SEQ ID NO: 9 GTCCACCTGTCCCTCCTGGGCTGCTGGATTGTCTCGTTTTCCTGCCAAATAAACAGGATCAGCGCTTTAC SEQ ID NO: 10GUCCACCUGUCCCUCCUGGGCUGCUGGAUUGUCUCGUUUUCCUGCCAAAU AAACAGGAUCAGCGCUUUAC

The term ‘a nucleic acid sequence, which is derived from the 3′UTR of a[ . . . ] gene’ preferably refers to a nucleic acid sequence, which isbased on the 3′UTR sequence of a [ . . . ] gene or on a part thereof,such as on the 3′UTR of an albumin gene, an α-globin gene, a β-globingene, a ribosomal protein gene, a tyrosine hydroxylase gene, alipoxygenase gene, or a collagen alpha gene, such as a collagen alpha1(l) gene, preferably of an albumin gene or on a part thereof. This termincludes sequences corresponding to the entire 3′UTR sequence, i.e. thefull length 3′UTR sequence of a gene, and sequences corresponding to afragment of the 3′UTR sequence of a gene, such as an albumin gene,α-globin gene, β-globin gene, ribosomal protein gene, tyrosinehydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as acollagen alpha 1(l) gene, preferably of an albumin gene.

The phrase “nucleic acid sequence, which is derived from the 3′UTR of a[ . . . ] gene” preferably refers to a nucleic acid sequence, which isbased on the 3′-UTR sequence of a [ . . . ] gene, preferably a genedescribed above, or on a fragment or part thereof. This phrase includessequences corresponding to the entire 3′-UTR sequence, i.e. the fulllength 3′-UTR sequence of said gene, and sequences corresponding to afragment of the 3′-UTR sequence of said gene. Preferably, a fragment ofa 3′-UTR of a [ . . . ] gene consists of a continuous stretch ofnucleotides corresponding to a continuous stretch of nucleotides in thefull-length 3′-UTR of a [ . . . ] gene, which represents at least 5%,10%, 20%, preferably at least 30%, more preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, even morepreferably at least 70%, even more preferably at least 80%, and mostpreferably at least 90% of the full-length 3′-UTR of a [ . . . ] gene.Such a fragment, in the sense of the present invention, is preferably afunctional fragment as described herein. Preferably, the fragmentretains a regulatory function for the translation of the ORF linked tothe 3′-UTR or fragment thereof. The term “3′-UTR of a [ . . . ] gene”preferably refers to the 3′-UTR of a naturally occurring gene,preferably as described herein.

The terms “variant of the 3′-UTR of a [ . . . ] gene” and “variantthereof”, in that context, refers to a variant of the 3′-UTR of anaturally occurring gene. Such variant may be a modified 3′-UTR of saidgene. For example, a variant 3′-UTR may exhibit one or more nucleotidedeletions, insertions, additions and/or substitutions compared to thenaturally occurring 3′-UTR, from which the variant is derived.Preferably, a variant of a 3′-UTR of a gene as used herein is at least40%, preferably at least 50%, more preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, even morepreferably at least 90%, most preferably at least 95% identical to thenaturally occurring 3′-UTR the variant is derived from. Preferably, thevariant is a functional variant as described herein.

The terms “functional variant”, “functional fragment”, and “functionalfragment of a variant” (also termed “functional variant fragment”) inthe context of the present invention, mean that the fragment of the3′-UTR of a gene, the variant of the 3′-UTR of a gene, or the fragmentof a variant of the 3′-UTR of a gene fulfils at least one, preferablymore than one, function of the naturally occurring 3′-UTR of therespective gene, of which the variant, the fragment, or the fragment ofa variant is derived. Such function may be, for example, stabilizingmRNA and/or enhancing, stabilizing and/or prolonging protein productionfrom an mRNA and/or increasing protein expression or total proteinproduction from an mRNA, preferably in a mammalian cell, such as in ahuman cell. Preferably, the function of the 3′-UTR of a gene asdescribed herein concerns the translation of the protein encoded by theORF. More preferably, the function comprises enhancing translationefficiency of the ORF linked to the 3′-UTR or fragment or variantthereof. It is particularly preferred that the variant, the fragment,and the variant fragment in the context of the present invention fulfilthe function of stabilizing an mRNA, preferably in a mammalian cell,such as a human cell, compared to an mRNA comprising a reference 3′-UTRor lacking a 3′-UTR, and/or the function of enhancing, stabilizingand/or prolonging protein production from an mRNA, preferably in amammalian cell, such as in a human cell, compared to an mRNA comprisinga reference 3′-UTR or lacking a 3′-UTR, and/or the function ofincreasing protein production from an mRNA, preferably in a mammaliancell, such as in a human cell, compared to an mRNA comprising areference 3′-UTR or lacking a 3′-UTR. A reference 3′-UTR may be, forexample, a 3′-UTR naturally occurring in combination with the ORF.Furthermore, a functional variant, a functional fragment, or afunctional variant fragment of a 3′-UTR of a gene as described hereinpreferably does not have a substantially diminishing effect on theefficiency of translation of the mRNA, which comprises such variant,fragment, or variant fragment of a 3′-UTR compared to the wild type3′-UTR, from which the variant, the fragment, or the variant fragment isderived. A particularly preferred function of a “functional fragment”, a“functional variant” or a “functional fragment of a variant” of the3′-UTR of a gene in the context of the present invention is theincrease, enhancement, stabilization and/or prolongation of proteinproduction by expression of an mRNA carrying the functional fragment,functional variant or functional fragment of a variant as describedabove.

Preferably, the efficiency of the one or more functions exerted by thefunctional variant, the functional fragment, or the functional variantfragment, such as mRNA and/or protein production stabilizing efficiencyand/or the protein production increasing efficiency, is increased by atleast 5%, more preferably by at least 10%, more preferably by at least20%, more preferably by at least 30%, more preferably by at least 40%,more preferably by at least 50%, more preferably by at least 60%, evenmore preferably by at least 70%, even more preferably by at least 80%,most preferably by at least 90% with respect to the mRNA and/or proteinproduction stabilizing efficiency and/or the protein productionincreasing efficiency exhibited by the naturally occurring 3′-UTR of therespective gene, from which the variant, the fragment or the variantfragment is derived.

In the context of the present invention, a fragment of the 3′-UTR of agene as described herein or of a variant of the 3′-UTR of such a genepreferably exhibits a length of at least about 3 nucleotides, preferablyof at least about 5 nucleotides, more preferably of at least about 10,15, 20, 25 or 30 nucleotides, even more preferably of at least about 50nucleotides, most preferably of at least about 70 nucleotides.Preferably, such fragment of the 3′-UTR of a gene or of a variant of the3′-UTR of a gene is a functional fragment as described above. In apreferred embodiment, the 3′-UTR of a gene or a fragment or variantthereof exhibits a length of between 3 and about 500 nucleotides,preferably of between 5 and about 150 nucleotides, more preferably ofbetween 10 and 100 nucleotides, even more preferably of between 15 and90, most preferably of between 20 and 70.

Preferably, the at least one 3′-UTR element of the artificial nucleicacid molecule according to the present invention comprises or consistsof a “functional fragment”, a “functional variant” or a “functionalfragment of a variant” of the 3′-UTR of a gene as described herein.

In a preferred embodiment, the at least one further 3′-UTR element,which may optionally be comprised in the 3′-UTR of the artificialnucleic acid molecule and which is distinct from a poly(A) sequence,increases the stability of the artificial nucleic acid molecule, e.g.increases the stability of an mRNA according to the present invention,compared to a respective nucleic acid (reference nucleic acid) lacking a3′-UTR or comprising a reference 3′-UTR lacking the optional 3′-UTRelement, such as a 3′-UTR naturally occurring in combination with theORF. Preferably, the at least one 3′-UTR element of the artificialnucleic acid molecule according to the present invention increases thestability of protein production from the artificial nucleic acidmolecule according to the present invention, e.g. from an mRNA accordingto the present invention, compared to a respective nucleic acid lackinga 3′-UTR or comprising a reference 3′-UTR, such as a 3′-UTR naturallyoccurring in combination with the ORF. Preferably, the at least one3′-UTR element of the artificial nucleic acid molecule according to thepresent invention prolongs protein production from the artificialnucleic acid molecule according to the present invention, e.g. from anmRNA according to the present invention, compared to a respectivenucleic acid lacking a 3′-UTR or comprising a reference 3′-UTR, such asa 3′-UTR naturally occurring in combination with the ORF. Preferably,the at least one 3′-UTR element of the artificial nucleic acid moleculeaccording to the present invention increases the protein expressionand/or total protein production from the artificial nucleic acidmolecule according to the present invention, e.g. from an mRNA accordingto the present invention, compared to a respective nucleic acid lackinga 3′-UTR or comprising a reference 3′-UTR, such as a 3′-UTR naturallyoccurring in combination with the ORF. Preferably, the at least one3′-UTR element of the artificial nucleic acid molecule according to thepresent invention does not negatively influence translational efficiencyof a nucleic acid compared to the translational efficiency of arespective nucleic acid lacking a 3′-UTR or comprising a reference3′-UTR, such as a 3′-UTR naturally occurring in combination with theORF. Even more preferably, the translation efficiency is enhanced by the3′-UTR in comparison to the translation efficiency of the proteinencoded by the respective ORF in its natural context.

The term “respective nucleic acid molecule” or “reference nucleic acidmolecule”, in this context, means that—apart from the different3′-UTRs—the reference nucleic acid molecule is comparable, preferablyidentical, to the inventive artificial nucleic acid molecule comprisingthe 3′-UTR element. In particular, a reference nucleic acid molecule maycomprise a nucleotide sequence and elements, such as ORF and 3′-UTR,which differs from the inventive artificial nucleic acid molecule onlyin the optional at least one 3′-UTR element, which is distinct from apoly(A) sequence.

The term “stabilizing and/or prolonging protein production” from anartificial nucleic acid molecule such as an artificial mRNA preferablymeans that the protein production from the artificial nucleic acidmolecule such as the artificial mRNA is stabilized and/or prolongedcompared to the protein production from a reference nucleic acidmolecule such as a reference mRNA, e.g. comprising a reference 3′-UTRlacking the 3′-UTR element or lacking a 3′-UTR altogether, preferably ina mammalian expression system, such as in HeLa or HDF cells. Thus,protein produced from the artificial nucleic acid molecule, such as theartificial mRNA, is observable for a longer period of time than what maybe seen for a protein produced from a reference nucleic acid molecule.In other words, the amount of protein produced from the artificialnucleic acid molecule such as the artificial mRNA measured over timeundercuts a threshold value at a later time point than the amount ofprotein produced from a reference nucleic acid molecule such as areference mRNA measured over time. Such a threshold value may be, forexample, the amount of protein measured in the initial phase ofexpression, such as 1, 2, 3, 4, 5 or 6 hours post initiation ofexpression, such as post transfection of the nucleic acid molecule.

For example, the protein production from the artificial nucleic acidmolecule, such as an artificial mRNA,—in an amount which is at least theamount observed in the initial phase of expression, such as 1, 2, 3, 4,5, or 6 hours post initiation of expression, such as post transfectionof the nucleic acid molecule—is prolonged by at least about 5 hours,preferably by at least about 10 hours, more preferably by at least about24 hours compared to the protein production from a reference nucleicacid molecule, such as a reference mRNA, in a mammalian expressionsystem, such as in mammalian cells, e.g. in HeLa or HDF cells. Thus, theartificial nucleic acid molecule according to the present inventionpreferably allows for prolonged protein production in an amount, whichis at least the amount observed in the initial phase of expression, suchas 1, 2, 3, 4, 5, or 6 hours post initiation of expression, such as posttransfection, by at least about 5 hours, preferably by at least about 10hours, more preferably by at least about 24 hours compared to areference nucleic acid molecule lacking a 3′-UTR or comprising areference 3′-UTR lacking the 3′-UTR element.

In preferred embodiments, the period of protein production from theartificial nucleic acid molecule according to the present invention isextended at least 1.5 fold, preferably at least 2 fold, more preferablyat least 2.5 fold compared to the protein production from a referencenucleic acid molecule lacking a 3′-UTR or comprising a reference 3′-UTRlacking the 3′-UTR element.

This effect of prolonging protein production may be determined by (i)measuring protein amounts, e.g. obtained by expression of an encodedreporter protein such as luciferase, preferably in a mammalianexpression system such as in HeLa or HDF cells, over time, (ii)determining the time point, at which the protein amount undercuts theamount of protein observed, e.g., at 1, 2, 3, 4, 5, or 6 hours postinitiation of expression, e.g. 1, 2, 3, 4, 5, or 6 hours posttransfection of the artificial nucleic acid molecule, and (iii)comparing the time point, at which the protein amount undercuts theprotein amount observed at 1, 2, 3, 4, 5, or 6 hours post initiation ofexpression to said time point determined for a nucleic acid moleculelacking a 3′-UTR or comprising a reference 3′-UTR lacking the 3′-UTRelement.

Preferably, this stabilizing and/or prolonging effect on proteinproduction is achieved, while the total amount of protein produced fromthe artificial nucleic acid molecule according to the present invention,e.g. within a time span of 48 or 72 hours, is at least the amount ofprotein produced from a reference nucleic acid molecule lacking a 3′-UTRor comprising a reference 3′-UTR lacking the 3′-UTR element, such as a3′-UTR naturally occurring with the ORF of the artificial nucleic acidmolecule. Thus, the present invention provides an artificial nucleicacid molecule, which allows for prolonged and/or stabilized proteinproduction in a mammalian expression system, such as in mammalian cells,e.g. in HeLa or HDF cells, as specified above, wherein the total amountof protein produced from said artificial nucleic acid molecule, e.g.within a time span of 48 or 72 hours, is at least the total amount ofprotein produced, e.g. within said time span, from a reference nucleicacid molecule lacking a 3′-UTR or comprising a reference 3′-UTR, such asa 3′-UTR naturally occurring with the ORF of the artificial nucleic acidmolecule.

Thus, “stabilized protein expression” preferably means that there ismore uniform protein production from the artificial nucleic acidmolecule according to the present invention over a predetermined periodof time, such as over 24 hours, more preferably over 48 hours, even morepreferably over 72 hours, when compared to a reference nucleic acidmolecule, for example, an mRNA comprising a reference 3′-UTR lacking the3′-UTR element or lacking a 3′-UTR altogether. Accordingly, the level ofprotein production, e.g. in a mammalian system, from the artificialnucleic acid molecule comprising a 3′-UTR element according to thepresent invention, e.g. from an mRNA according to the present invention,preferably does not drop to the extent observed for a reference nucleicacid molecule, such as a reference mRNA as described above. For example,the amount of a protein (encoded by the ORF) observed 6 hours afterinitiation of expression, e.g. 6 hours post transfection of theartificial nucleic acid molecule according to the present invention intoa cell, such as a mammalian cell, may be comparable to the amount ofprotein observed 48 hours after initiation of expression, e.g. 48 hourspost transfection. Thus, the ratio of the amount of protein encoded bythe ORF, such as of a reporter protein, e.g., luciferase, observed at 48hours post initiation of expression, e.g. 48 hours post transfection, tothe amount of protein observed 6 hours after initiation of expression,e.g. 6 hours post transfection, is preferably at least about 0.4, morepreferably at least about 0.5, more preferably at least about 0.6, evenmore preferably at least about 0.7. Preferably, the ratio is betweenabout 0.4 and about 4, preferably between about 0.65 and about 3, morepreferably between about 0.7 and 2 for a nucleic acid molecule accordingto the present invention. For a respective reference nucleic acidmolecule, e.g. an mRNA comprising a reference 3′-UTR lacking the 3′-UTRelement or lacking a 3′-UTR altogether, said ratio may be, e.g. betweenabout 0.05 and about 0.3.

Thus, in a preferred embodiment, the present invention provides anartificial nucleic acid molecule comprising an ORF and a 3′-UTRcomprising an optional 3′-UTR element as described above, wherein theratio of the (reporter) protein amount, e.g. the amount of luciferase,observed 48 hours after initiation of expression to the (reporter)protein amount observed 6 hours after initiation of expression,preferably in a mammalian expression system, such as in mammalian cells,e.g. in HeLa cells, is preferably above about 0.4, more preferably aboveabout 0.5, more preferably above about 0.6, even more preferably aboveabout 0.7, e.g. between about 0.4 and about 4, preferably between about0.65 and about 3, more preferably between about 0.7 and 2, whereinpreferably the total amount of protein produced from said artificialnucleic acid molecule, e.g. within a time span of 48 hours, is at leastthe total amount of protein produced, e.g. within said time span, from areference nucleic acid molecule lacking a 3′-UTR or comprising areference 3′-UTR lacking the optional 3′-UTR element, such as a 3′-UTRnaturally occurring with the ORF of the artificial nucleic acidmolecule. In a preferred embodiment, the present invention provides anartificial nucleic acid molecule comprising an ORF and a 3′-UTRcomprising an optional 3′-UTR element as described above, wherein theratio of the (reporter) protein amount, e.g. the amount of luciferase,observed 72 hours after initiation of expression to the (reporter)protein amount observed 6 hours after initiation of expression,preferably in a mammalian expression system, such as in mammalian cells,e.g. in HeLa cells, is preferably above about 0.4, more preferably aboveabout 0.5, more preferably above about 0.6, even more preferably aboveabout 0.7, e.g. between about 0.4 and 1.5, preferably between about 0.65and about 1.15, more preferably between about 0.7 and 1.0, whereinpreferably the total amount of protein produced from said artificialnucleic acid molecule, e.g. within a time span of 72 hours, is at leastthe total amount of protein produced, e.g. within said time span, from areference nucleic acid molecule lacking a 3′-UTR or comprising areference 3′-UTR lacking the optional 3′-UTR element, such as a 3′-UTRnaturally occurring with the ORF of the artificial nucleic acidmolecule.

In a preferred embodiment, the at least one further 3′-UTR element,which may optionally be comprised in the 3′-UTR of the artificialnucleic acid molecule and which is distinct from a poly(A) sequence,increases or enhances the protein expression from the artificial nucleicacid molecule as defined herein.

Said increase in stability of the artificial nucleic acid molecule, saidincrease in stability of protein production, said prolongation ofprotein production and/or said increase/enhancement in proteinexpression and/or total protein production is preferably determined bycomparison with a respective reference nucleic acid molecule lacking a3′-UTR, e.g. an mRNA lacking a 3′-UTR, or a reference nucleic acidmolecule comprising a reference 3′-UTR lacking the optional 3′-UTRelement, such as a 3′-UTR naturally occurring with the ORF as describeabove.

The mRNA and/or protein production stabilizing effect and efficiencyand/or the protein production increasing effect and efficiency of the atleast one (optional) 3′-UTR element of the artificial nucleic acidmolecule according to the present invention may be determined by anymethod suitable for this purpose known to skilled person. For example,artificial mRNA molecules may be generated comprising a codingsequence/open reading frame (ORF) for a reporter protein, such asluciferase, and no 3′-UTR, a 3′-UTR lacking the optional 3′-UTR element,such as a 3′-UTR derived from a naturally occurring gene or a 3′-UTRderived from a reference gene (i.e., a reference 3′-UTR, such as a3′-UTR naturally occurring with the ORF). Such mRNAs may be generated,for example, by in vitro transcription of respective vectors such asplasmid vectors, e.g. comprising a T7 promoter and a sequence encodingthe respective mRNA sequences. The generated mRNA molecules may betransfected into cells by any transfection method suitable fortransfecting mRNA, for example they may be electroporated into mammaliancells, such as HELA cells, and samples may be analyzed certain timepoints after transfection, for example, 6 hours, 24 hours, 48 hours, and72 hours post transfection. Said samples may be analyzed for mRNAquantities and/or protein quantities by methods well known to theskilled person. For example, the quantities of reporter mRNA present inthe cells at the sample time points may be determined by quantitativePCR methods. The quantities of reporter protein encoded by therespective mRNAs may be determined, e.g., by Western Blot, ELISA assays,FACS analysisor reporter assays such as luciferase assays depending onthe reporter protein used. The effect of stabilizing protein expressionand/or prolonging protein expression may be, for example, analyzed bydetermining the ratio of the protein level observed 48 hours posttransfection and the protein level observed 6 hours post transfection.The closer said value is to 1, the more stable the protein expression iswithin this time period. Such measurements may of course also beperformed at 72 or more hours and the ratio of the protein levelobserved 72 hours post transfection and the protein level observed 6hours post transfection may be determined to determine stability ofprotein expression.

In some embodiments, the 3′-UTR of the artificial nucleic acid moleculemay comprise a histone stem-loop in addition to the at least one poly(A)sequence and the optional 3′-UTR element as described herein. Such3′-UTR of the artificial nucleic acid molecule according to the presentinvention may comprise, for example, in 5′-to-3′-direction, an optional3′-UTR element as described herein, at least one poly(A) sequence, anoptional poly(C) sequence, an optional histone stem-loop sequence, andoptionally a further poly(A) sequence or a polyadenylation signal.

In a preferred embodiment, the artificial nucleic acid moleculeaccording to the invention comprises at least one histone stem-loopsequence.

Such histone stem-loop sequences are preferably selected from histonestem-loop sequences as disclosed in WO 2012/019780, whose disclosure isincorporated herewith by reference.

A histone stem-loop sequence, suitable to be used within the presentinvention, is preferably selected from at least one of the followingformulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

stem1 or stem2 is a consecutive sequence of 1 to 6, preferably of 2 tobordering 6, more preferably of 2 to 5, even more preferably of elementN₁₋₆ 3 to 5, most preferably of 4 to 5 or 5N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C, or a nucleotide analogue thereof; stem1 [N₀₋₂GN₃₋₅] isreverse complementary or partially reverse complementary with elementstem2, and is a consecutive sequence between of 5 to 7 nucleotides;wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1,more preferably of 1N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof; wherein N₃₋₅ is a consecutive sequence of 3to 5, preferably of 4 to 5, more preferably of 4N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or a nucleotide analogue thereof, and wherein G isguanosine or an analogue thereof, and may be optionally replaced by acytidine or an analogue thereof, provided that its complementarynucleotide cytidine in stem2 is replaced by guanosine; loop sequence islocated between elements stem1 and stem2, and [N₀₋₄(U/T)N₀₋₄] is aconsecutive sequence of 3 to 5 nucleotides, more preferably of 4nucleotides; wherein each N₀₋₄ is independent from another a consecutivesequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2N,wherein each N is independently from another selected from a nucleotideselected from A, U, T, G and C or a nucleotide analogue thereof; andwherein U/T represents uridine, or optionally thymidine; stem2[N₃₋₅CN₀₋₂] is reverse complementary or partially reverse complementarywith element stem1, and is a consecutive sequence between of 5 to 7nucleotides; wherein N₃₋₅ is a consecutive sequence of 3 to 5,preferably of 4 to 5, more preferably of 4N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or a nucleotide analogue thereof; wherein N₀₋₂ is aconsecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of1N, wherein each N is independently from another selected from anucleotide selected from A, U, T, G or C or a nucleotide analoguethereof; and wherein C is cytidine or an analogue thereof, and may beoptionally replaced by a guanosine or an analogue thereof provided thatits complementary nucleoside guanosine in stem1 is replaced by cytidine;

wherein

stem1 and stem2 are capable of base pairing with each other forming areverse complementary sequence, wherein base pairing may occur betweenstem1 and stem2, e.g. by Watson-Crick base pairing of nucleotides A andU/T or G and C or by non-Watson-Crick base pairing e.g. wobble basepairing, reverse Watson-Crick base pairing, Hoogsteen base pairing,reverse Hoogsteen base pairing or are capable of base pairing with eachother forming a partially reverse complementary sequence, wherein anincomplete base pairing may occur between stem1 and stem2, on the basisthat one ore more bases in one stem do not have a complementary base inthe reverse complementary sequence of the other stem.

According to a further preferred embodiment, the histone stem-loopsequence may be selected according to at least one of the followingspecific formulae (Ia) or (IIa):

formula (Ia) (stem-loop sequence without stem bordering elements):

formula (IIa) (stem-loop sequence with stem bordering elements):

wherein:

N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of thefirst aspect, the artificial nucleic acid molecule sequence may compriseat least one histone stem-loop sequence according to at least one of thefollowing specific formulae (Ib) or (IIb):

formula (Ib) (stem-loop sequence without stem bordering elements):

formula (IIb) (stem-loop sequence with stem bordering elements):

wherein:

N, C, G, T and U are as defined above.

A particular preferred histone stem-loop sequence is the sequenceaccording to SEQ ID NO: 11: CAAAGGCTCTTTTCAGAGCCACCA or more preferablythe corresponding RNA sequence of the nucleic acid sequence according toSEQ ID NO: 11.

In some embodiments, the artificial nucleic acid molecule comprisesfurther elements such as a 5′-cap, a poly(C) sequence and/or anIRES-motif. A 5′-cap may be added to the 5′end of an RNA duringtranscription or post-transcriptionally. Furthermore, the inventiveartificial nucleic acid molecule, particularly if the nucleic acid is inthe form of an mRNA or encodes an mRNA, may be modified by a sequence ofat least 10 cytidines, preferably at least 20 cytidines, more preferablyat least 30 cytidines (so-called “poly(C) sequence”). In particular, theinventive artificial nucleic acid molecule may contain, especially ifthe nucleic acid is in the form of an (m)RNA or encodes an mRNA, apoly(C) sequence of typically about 10 to 200 cytidine nucleotides,preferably about 10 to 100 cytidine nucleotides, more preferably about10 to 70 cytidine nucleotides or even more preferably about 20 to 50 oreven 20 to 30 cytidine nucleotides. Most preferably, the inventivenucleic acid comprises a poly(C) sequence of 30 cytidine residues. Thus,preferably the artificial nucleic acid molecule according to the presentinvention comprises, preferably in 5′-to-3′ direction, an ORF, at leastone 3′-UTR element as described above, at least one poly(A) sequence, apoly(C) sequence, a histone stem-loop sequence and, optionally, afurther poly(A) sequence.

An internal ribosome entry site (IRES) sequence or IRES-motif mayseparate several open reading frames, for example if the artificialnucleic acid molecule encodes two or more peptides or proteins. AnIRES-sequence may be particularly helpful if the artificial nucleic acidmolecule is a bi- or multicistronic nucleic acid molecule.

Furthermore, the artificial nucleic acid molecule may compriseadditional 5′-elements, preferably a 5′-UTR, a promoter, or a 5′-UTR anda promoter containing-sequence. The promoter may drive and/or regulatetranscription of the artificial nucleic acid molecule according to thepresent invention, for example of an artificial DNA-molecule accordingto the present invention. Furthermore, the 5′-UTR may consist of or maycomprise the 5′-UTR of a gene as defined herein. Furthermore, the 5′-UTRmay interact with the 3′-UTR of the inventive artifical nucleic acidmolecule and thus may support the effect of the 3′-UTR of the inventivenucleic acid molecule. Such elements may further support stability andtranslational efficiency.

In a particularly preferred embodiment of the present invention, theartificial nucleic acid molecule comprises at least one 5′-untranslatedregion element (5′UTR element), which comprises or consists of a nucleicacid sequence, which is derived from the 5′UTR of a TOP gene or which isderived from a fragment, homolog or variant of the 5′UTR of a TOP gene.

It is particularly preferred that the 5′UTR element does not comprise aTOP-motif or a 5′TOP, as defined above.

The nucleic acid sequence, which is derived from the 5′UTR of a TOPgene, is preferably derived from a eukaryotic TOP gene, preferably aplant or animal TOP gene, more preferably a chordate TOP gene, even morepreferably a vertebrate TOP gene, most preferably a mammalian TOP gene,such as a human TOP gene.

For example, the 5′UTR element is preferably selected from 5′-UTRelements comprising or consisting of a nucleic acid sequence, which isderived from a nucleic acid sequence selected from the group consistingof SEQ ID NOs. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.1422 of the patent application WO2013/143700, whose disclosure isincorporated herein by reference, from the homologs of SEQ ID NOs.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700, from a variant thereof, or preferablyfrom a corresponding RNA sequence. The term “homologs of SEQ ID NOs.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700” refers to sequences of other speciesthan Homo sapiens, which are homologous to the sequences according toSEQ ID NOs. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422of the patent application WO2013/143700.

In a preferred embodiment, the 5′UTR element comprises or consists of anucleic acid sequence, which is derived from a nucleic acid sequenceextending from nucleotide position 5 (i.e. the nucleotide that islocated at position 5 in the sequence) to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID NOs. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID NOs. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence. It is particularly preferred that the 5′ UTR element isderived from a nucleic acid sequence extending from the nucleotideposition immediately 3′ to the 5′TOP to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID NOs. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID NOs. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence.

In a particularly preferred embodiment, the 5′UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 5′UTR of aTOP gene encoding a ribosomal protein or from a variant of a 5′UTR of aTOP gene encoding a ribosomal protein. For example, the 5′UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 5′UTR of a nucleic acid sequence according to any of SEQ ID NOs: 170,232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292,1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304,1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316,1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328,1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340,1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353,1354, 1355, 1356, 1357, 1358, 1359, or 1360 of the patent applicationWO2013/143700, a corresponding RNA sequence, a homolog thereof, or avariant thereof as described herein, preferably lacking the 5′TOP motif.As described above, the sequence extending from position 5 to thenucleotide immediately 5′ to the ATG (which is located at the 3′end ofthe sequences) corresponds to the 5′UTR of said sequences.

Preferably, the 5′UTR element comprises or consists of a nucleic acidsequence, which is derived from a 5′UTR of a TOP gene encoding aribosomal Large protein (RPL) or from a homolog or variant of a 5′UTR ofa TOP gene encoding a ribosomal Large protein (RPL). For example, the5′UTR element comprises or consists of a nucleic acid sequence, which isderived from a 5′UTR of a nucleic acid sequence according to any of SEQID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and1422 of the patent application WO2013/143700, a corresponding RNAsequence, a homolog thereof, or a variant thereof as described herein,preferably lacking the 5′TOP motif.

In a particularly preferred embodiment, the 5′UTR element comprises orconsists of a nucleic acid sequence, which is derived from the 5′UTR ofa ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, or from a variant of the 5′UTR ofa ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, wherein preferably the 5′UTRelement does not comprise the 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′UTR elementcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 12 (5′-UTR ofhuman ribosomal protein Large 32 lacking the 5′ terminal oligopyrimidinetract: GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC; corresponding to SEQID No. 1368 of the patent application WO2013/143700) or preferably to acorresponding RNA sequence, or wherein the at least one 5′UTR elementcomprises or consists of a fragment of a nucleic acid sequence, whichhas an identity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID No. 12or more preferably to a corresponding RNA sequence, wherein, preferably,the fragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20%, preferably at least 30%, morepreferably at least 40% of the full-length 5′UTR. Preferably, thefragment exhibits a length of at least about 20 nucleotides or more,preferably of at least about 30 nucleotides or more, more preferably ofat least about 40 nucleotides or more. Preferably, the fragment is afunctional fragment as described herein.

In some embodiments, the artificial nucleic acid molecule comprises a5′UTR element, which comprises or consists of a nucleic acid sequence,which is derived from the 5′UTR of a vertebrate TOP gene, such as amammalian, e.g. a human TOP gene, selected from RPSA, RPS2, RPS3, RPS3A,RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14,RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24,RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5,RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13,RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23,RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32,RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40,RPL41, RPLP0, RPLP1, RPLP2, RPLP3, RPLP0, RPLP1, RPLP2, EEF1A1, EEF1B2,EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP,EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2,UQCRB or from a homolog or variant thereof, wherein preferably the 5′UTRelement does not comprise a TOP-motif or the 5′TOP of said genes, andwherein optionally the 5′UTR element starts at its 5′-end with anucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10downstream of the 5′terminal oligopyrimidine tract (TOP) and whereinfurther optionally the 5′UTR element, which is derived from a 5′UTR of aTOP gene, terminates at its 3′-end with a nucleotide located at position1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (A(U/T)G) ofthe gene it is derived from.

Accordingly, in some embodiments, the invention provides artificialnucleic acid molecules, preferably mRNA molecules, comprising in5′-to-3′-direction at least one of the following structures

ORF-poly(A) sequence;

ORF-poly(A) sequence-poly(A) sequence;

ORF-IRES-ORF-poly(A) sequence;

ORF-3′-UTR element-poly(A) sequence;

ORF-poly(A) sequence-3′-UTR element;

ORF-3′-UTR element-poly(A) sequence-poly(C) sequence-histone stem-loop;

ORF-3′-UTR element-poly(A) sequence-poly(C) sequence-poly(A) sequence;

ORF-3′-UTR element-poly(A) sequence-histone stem-loop-poly(A) sequence;

ORF-3′-UTR element-poly(A) sequence-poly(C) sequence-histonestem-loop-poly(A) sequence;

5′-UTR-ORF-3′-UTR element-poly(A) sequence-poly(C) sequence-histonestem-loop-poly(A) sequence; or

5′-cap-5′-UTR-ORF-3′-UTR element-poly(A) sequence-poly(C)sequence-histone stem-loop-poly(A) sequence.

Preferably, the artificial nucleic acid molecule according to thepresent invention, preferably the open reading frame thereof, is atleast partially G/C modified. Thus, the inventive artificial nucleicacid molecule may be thermodynamically stabilized by modifying the G(guanosine) and/or C (cytidine) content (G/C content) of the molecule.The G/C content of the open reading frame of an artificial nucleic acidmolecule according to the present invention may be increased compared tothe G/C content of the open reading frame of a corresponding wild typesequence, preferably by using the degeneration of the genetic code.Thus, the encoded amino acid sequence of the artificial nucleic acidmolecule is preferably not modified by the G/C modification compared tothe coded amino acid sequence of the particular wild type sequence. Thecodons of the coding sequence or the whole artificial nucleic acidmolecule, e.g. an mRNA, may therefore be varied compared to the wildtype coding sequence, such that they include an increased amount of G/Cnucleotides while the translated amino acid sequence is maintained. Dueto the fact that several codons encode one and the same amino acid(so-called degeneration of the genetic code), it is feasible to altercodons while not altering the encoded peptide/protein sequence(so-called alternative codon usage). Hence, it is possible tospecifically introduce certain codons (in exchange for the respectivewild-type codons encoding the same amino acid), which are morefavourable with respect to stability of RNA and/or with respect to codonusage in a subject (so-called codon optimization).

Depending on the amino acid to be encoded by the coding region of theinventive artificial nucleic acid molecule as defined herein, there arevarious possibilities for modification of the nucleic acid sequence,e.g. the open reading frame, compared to its wild type coding region. Inthe case of amino acids, which are encoded by codons, which containexclusively G or C nucleotides, no modification of the codon isnecessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala(GCC or GCG) and Gly (GGC or GGG) require no modification, since no A orU/T is present.

In contrast, codons, which contain A and/or U/T nucleotides may bemodified by substitution of other codons, which encode the same aminoacids but contain no A and/or U/T. For example

the codons for Pro can be modified from CC(U/T) or CCA to CCC or CCG;

the codons for Arg can be modified from CG(U/T) or CGA or AGA or AGG toCGC or CGG;

the codons for Ala can be modified from GC(U/T) or GCA to GCC or GCG;

the codons for Gly can be modified from GG(U/T) or GGA to GGC or GGG.

In other cases, although A or (U/T) nucleotides cannot be eliminatedfrom the codons, it is however possible to decrease the A and (U/T)content by using codons, which contain a lower content of A and/or (U/T)nucleotides. Examples of these are:

The codons for Phe can be modified from (U/T)(U/T)(U/T) to (U/T) (U/T)C;

the codons for Leu can be modified from (U/T) (U/T)A, (U/T) (U/T)G,C(U/T) (U/T) or C(U/T)A to C(U/T)C or C(U/T)G;

the codons for Ser can be modified from (U/T)C(U/T) or (U/T)CA orAG(U/T) to (U/T)CC, (U/T)CG or AGC;

the codon for Tyr can be modified from (U/T)A(U/T) to (U/T)AC;

the codon for Cys can be modified from (U/T)G(U/T) to (U/T)GC;

the codon for His can be modified from CA(U/T) to CAC;

the codon for Gln can be modified from CAA to CAG;

the codons for Ile can be modified from A(U/T)(U/T) or A(U/T)A toA(U/T)C;

the codons for Thr can be modified from AC(U/T) or ACA to ACC or ACG;

the codon for Asn can be modified from AA(U/T) to AAC;

the codon for Lys can be modified from AAA to AAG;

the codons for Val can be modified from G(U/T)(U/T) or G(U/T)A toG(U/T)C or G(U/T)G;

the codon for Asp can be modified from GA(U/T) to GAC;

the codon for Glu can be modified from GAA to GAG;

the stop codon (U/T)AA can be modified to (U/T)AG or (U/T)GA.

In the case of the codons for Met (A(U/T)G) and Trp ((U/T)GG), on theother hand, there is no possibility of sequence modification withoutaltering the encoded amino acid sequence.

The substitutions listed above can be used either individually or in allpossible combinations to increase the G/C content of the open readingframe of the inventive artificial nucleic acid molecule as definedherein, compared to its particular wild type open reading frame (i.e.the original sequence). Thus, for example, all codons for Thr occurringin the wild type sequence can be modified to ACC (or ACG).

Preferably, the G/C content of the open reading frame of the inventiveartificial nucleic acid molecule as defined herein is increased by atleast 7%, more preferably by at least 15%, particularly preferably by atleast 20%, compared to the G/C content of the wild type coding regionwithout altering the encoded amino acid sequence, i.e. using thedegeneracy of the genetic code. According to a specific embodiment atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%,even more preferably at least 80% and most preferably at least 90%, 95%or even 100% of the substitutable codons in the open reading frame ofthe inventive artificial nucleic acid molecule or a fragment, variant orderivative thereof are substituted, thereby increasing the G/C contentof said open reading frame.

In this context, it is particularly preferable to increase the G/Ccontent of the open reading frame of the inventive artificial nucleicacid molecule as defined herein, to the maximum (i.e. 100% of thesubstitutable codons), compared to the wild type open reading frame,without altering the encoded amino acid sequence.

Furthermore, the open reading frame is preferably at least partiallycodon-optimized. Codon-optimization is based on the finding that thetranslation efficiency may be determined by a different frequency in theoccurrence of transfer RNAs (tRNAs) in cells. Thus, if so-called “rarecodons” are present in the coding region of the inventive artificialnucleic acid molecule as defined herein, to an increased extent, thetranslation of the corresponding modified nucleic acid sequence is lessefficient than in the case, where codons coding for relatively“frequent” tRNAs are present.

Thus, the open reading frame of the inventive artificial nucleic acidmolecule is preferably modified compared to the corresponding wild typecoding region such that at least one codon of the wild type sequence,which is recognized by a tRNA, which is relatively rare in the cell, isexchanged for a codon, which is recognized by a tRNA, which iscomparably frequent in the cell and carries the same amino acid as therelatively rare tRNA. By this modification, the open reading frame ofthe inventive artificial nucleic acid molecule as defined herein, ismodified such that codons, for which frequently occurring tRNAs areavailable, may replace codons, which correspond to rare tRNAs. In otherwords, according to the invention, by such a modification all codons ofthe wild type open reading frame, which are recognized by a rare tRNA,may be exchanged for a codon, which is recognized by a tRNA, which ismore frequent in the cell and which carries the same amino acid as therare tRNA. Which tRNAs occur relatively frequently in the cell andwhich, in contrast, occur relatively rarely, is known to a personskilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001,11(6): 660-666. Accordingly, preferably, the open reading frame iscodon-optimized, preferably with respect to the system, in which theartificial nucleic acid molecule according to the present invention isto be expressed, preferably with respect to the system, in which theartificial nucleic acid molecule according to the present invention isto be translated. Preferably, the codon usage of the open reading frameis codon-optimized according to mammalian codon usage, more preferablyaccording to human codon usage. Preferably, the open reading frame iscodon-optimized and G/C-content modified.

For further improving degradation resistance, e.g. resistance to in vivodegradation by an exo- or endonuclease, and/or for further improvingstability of protein expression from the artificial nucleic acidmolecule according to the present invention, the artificial nucleic acidmolecule may further comprise modifications, such as backbonemodifications, sugar modifications and/or base modifications, e.g.,lipid-modifications or the like. Preferably, the transcription and/orthe translation of the artificial nucleic acid molecule according to thepresent invention is not significantly impaired by said modifications.

Generally, the artificial nucleic acid molecule of the present inventionmay comprise any native (=naturally occurring) nucleotide, e.g.guanosine, uracil, adenosine, and/or cytosine or an analogue thereof. Inthis respect, nucleotide analogues are defined as natively andnon-natively occurring variants of the naturally occurring nucleotidesadenosine, cytosine, thymidine, guanosine and uridine. Accordingly,analogues are e.g. chemically derivatized nucleotides with non-nativelyoccurring functional groups, which are preferably added to or deletedfrom the naturally occurring nucleotide or which substitute thenaturally occurring functional groups of a nucleotide. Accordingly, eachcomponent of the naturally occurring nucleotide may be modified, namelythe base component, the sugar (ribose) component and/or the phosphatecomponent forming the backbone (see above) of the RNA sequence.Analogues of guanosine, uridine, adenosine, thymidine and cytosineinclude, without implying any limitation, any natively occurring ornon-natively occurring guanosine, uridine, adenosine, thymidine orcytosine that has been altered e.g. chemically, for example byacetylation, methylation, hydroxylation, etc., including1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine,2,2-dimethyl-guanosine, 2,6-diaminopurine, 2′-Amino-2′-deoxyadenosine,2′-Amino-2′-deoxycytidine, 2′-Amino-2′-deoxyguanosine,2′-Amino-2′-deoxyuridine, 2-Amino-6-chloropurineriboside,2-Aminopurine-riboside, 2′-Araadenosine, 2′-Aracytidine, 2′-Arauridine,2′-Azido-2′-deoxyadenosine, 2′-Azido-2′-deoxycytidine,2′-Azido-2′-deoxyguanosine, 2′-Azido-2′-deoxyuridine, 2-Chloroadenosine,2′-Fluoro-2′-deoxyadenosine, 2′-Fluoro-2′-deoxycytidine,2′-Fluoro-2′-deoxyguanosine, 2′-Fluoro-2′-deoxyuridine,2′-Fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine,2-methyl-thio-N6-isopenenyl-adenosine, 2′-O-Methyl-2-aminoadenosine,2′-O-Methyl-2′-deoxyadenosine, 2′-O-Methyl-2′-deoxycytidine,2′-O-Methyl-2′-deoxyguanosine, 2′-O-Methyl-2′-deoxyuridine,2′-O-Methyl-5-methyluridine, 2′-O-Methylinosine,2′-O-Methylpseudouridine, 2-Thiocytidine, 2-thio-cytosine,3-methyl-cytosine, 4-acetyl-cytosine, 4-Thiouridine,5-(carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine,5-Aminoallylcytidine, 5-Aminoallyl-deoxy-uridine, 5-Bromouridine,5-carboxymehtylaminomethyl-2-thio-uracil,5-carboxymethylamonomethyl-uracil, 5-Chloro-Ara-cytosine,5-Fluoro-uridine, 5-lodouridine, 5-methoxycarbonylmethyl-uridine,5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-Azauridine,6-Chloro-7-deaza-guanosine, 6-Chloropurineriboside,6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside,7-Deaza-2′-deoxy-guanosine, 7-Deazaadenosine, 7-methyl-guanosine,8-Azaadenosine, 8-Bromo-adenosine, 8-Bromo-guanosine,8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazole-riboside,Beta-D-mannosyl-queosine, Dihydro-uracil, Inosine, N1-Methyladenosine,N6-([6-Aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine,N6-methyl-adenosine, N7-Methyl-xanthosine, N-uracil-5-oxyacetic acidmethyl ester, Puromycin, Queosine, Uracil-5-oxyacetic acid,Uracil-5-oxyacetic acid methyl ester, Wybutoxosine, Xanthosine, andXylo-adenosine. The preparation of such analogues is known to a personskilled in the art, for example from U.S. Pat. Nos. 4,373,071,4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679,5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642. In the case ofan analogue as described above, particular preference may be givenaccording to certain embodiments of the invention to those analoguesthat increase the protein expression of the encoded peptide or proteinor that increase the immunogenicity of the artificial nucleic acidmolecule of the invention and/or do not interfere with a furthermodification of the artificial nucleic acid molecule that has beenintroduced.

According to a particular embodiment, the artificial nucleic acidmolecule of the present invention can contain a lipid modification.

In a preferred embodiment, the artificial nucleic acid moleculeaccording to the invention comprises, preferably from 5′ to 3′direction, the following elements:

an ORF;

an optional 3′-UTR element as described herein;

a poly(A) sequence, preferably comprising or consisting of 64 adenosineresidues;

a poly(C) sequence, preferably comprising or consisting of 30 cytosineresidues;

a histone stem-loop sequence, preferably comprising or consisting of thenucleic acid sequence according to SEQ ID NO: 11; and

a poly(A) sequence, preferably comprising at least 160 adenosineresidues.

Preferably, the artificial nucleic acid molecule, which is preferably anmRNA, optionally further comprises a 5′-UTR, preferably comprising a5′-UTR element comprising or consisting of a nucleic acid, which isderived from a 5′-UTR of a TOP gene encoding a ribosomal Large protein(RPL) or from a homolog, a fragment or variant thereof, preferablylacking the 5′ TOP motif. Even more preferably, the inventive artificialnucleic acid molecule further comprises a 5′-cap structure.

In a preferred embodiment, the at least one open reading frame of theinventive artificial nucleic acid molecule encodes a therapeutic proteinor peptide. In another embodiment, an antigen is encoded by the at leastone open reading frame, such as a pathogenic antigen, a tumour antigen,an allergenic antigen or an autoimmune antigen. Therein, theadministration of the artificial nucleic acid molecule encoding theantigen is used in a genetic vaccination approach against a diseaseinvolving said antigen.

In an alternative embodiment, an antibody is encoded by the at least oneopen reading frame of the artificial nucleic acid molecule according tothe invention.

Antigens:

Pathogenic Antigens:

The artificial nucleic acid molecule according to the present inventionmay encode a protein or a peptide, which comprises a pathogenic antigenor a fragment, variant or derivative thereof. Such pathogenic antigensare derived from pathogenic organisms, in particular bacterial, viral orprotozoological (multicellular) pathogenic organisms, which evoke animmunological reaction in a subject, in particular a mammalian subject,more particularly a human. More specifically, pathogenic antigens arepreferably surface antigens, e.g. proteins (or fragments of proteins,e.g. the exterior portion of a surface antigen) located at the surfaceof the virus or the bacterial or protozoological organism.

Pathogenic antigens are peptide or protein antigens preferably derivedfrom a pathogen associated with infectious disease which are preferablyselected from antigens derived from the pathogens Acinetobacterbaumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostomabraziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum,Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus,Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus,Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis,Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus,Brugia malayi, Bunyaviridae family, Burkholderia cepacia and otherBurkholderia species, Burkholderia mallei, Burkholderia pseudomallei,Caliciviridae family, Campylobacter genus, Candida albicans, Candidaspp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophilapsittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium perfringens,Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus,Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4),Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichiachaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica,Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie Avirus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus(EBV), Escherichia coli O157:H7, O111 and O104:H4, Fasciola hepatica andFasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,Francisella tularensis, Fusobacterium genus, Geotrichum candidum,Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis BVirus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis EVirus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasmacapsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Humanbocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7(HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus,Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassavirus, Legionella pneumophila, Leishmania genus, Leptospira genus,Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV),Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Naegleria fowleri, Necator americanus, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp,Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family(Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimuswestermani, Parvovirus B19, Pasteurella genus, Plasmodium genus,Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytialvirus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsiagenus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi,Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus,Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosomagenus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrixschenckii, Staphylococcus genus, Staphylococcus genus, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes,Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borneencephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasmagondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis,Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosomacruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicellazoster virus (VZV), Variola major or Variola minor, vCJD prion,Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus,Western equine encephalitis virus, Wuchereria bancrofti, Yellow fevervirus, Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis.

In this context, particularly preferred are antigens from the pathogensselected from Influenza virus, respiratory syncytial virus (RSV), Herpessimplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiencyvirus (HIV), Plasmodium, Staphylococcus aureus, Dengue virus, Chlamydiatrachomatis, Cytomegalovirus (CMV), Hepatitis B virus (HBV),Mycobacterium tuberculosis, Rabies virus, and Yellow Fever Virus.

Tumour Antigens:

In a further embodiment the artificial nucleic acid molecule accordingto the present invention may encode a protein or a peptide, whichcomprises a peptide or protein comprising a tumour antigen, a fragment,variant or derivative of said tumour antigen, preferably, wherein thetumour antigen is a melanocyte-specific antigen, a cancer-testis antigenor a tumour-specific antigen, preferably a CT-X antigen, a non-XCT-antigen, a binding partner for a CT-X antigen or a binding partnerfor a non-X CT-antigen or a tumour-specific antigen, more preferably aCT-X antigen, a binding partner for a non-X CT-antigen or atumour-specific antigen or a fragment, variant or derivative of saidtumour antigen; and wherein each of the nucleic acid sequences encodes adifferent peptide or protein; and wherein at least one of the nucleicacid sequences encodes for 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m,alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1,BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsinB, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55,CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66,COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m,EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2,FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin,Her2/neu, HERV-K-MEL, HLA-A*0201-R171, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin classl/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE,PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1,survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP,TEL-AML1, TGFbeta, TGFbetaRll, TGM-4, TPI/m, TRAG-3, TRG, TRP-1,TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, WT1and a immunoglobulin idiotype of a lymphoid blood cell or a T cellreceptor idiotype of a lymphoid blood cell, or a fragment, variant orderivative of said tumour antigen; preferably survivin or a homologuethereof, an antigen from the MAGE-family or a binding partner thereof ora fragment, variant or derivative of said tumour antigen. Particularlypreferred in this context are the tumour antigens NY-ESO-1, 5T4,MAGE-C1, MAGE-C2, Survivin, Muc-1, PSA, PSMA, PSCA, STEAP and PAP.

In a preferred embodiment, the artificial nucleic acid molecule encodesa protein or a peptide, which comprises a therapeutic protein or afragment, variant or derivative thereof.

Therapeutic proteins as defined herein are peptides or proteins, whichare beneficial for the treatment of any inherited or acquired disease orwhich improves the condition of an individual. Particularly, therapeuticproteins play an important role in the creation of therapeutic agentsthat could modify and repair genetic errors, destroy cancer cells orpathogen infected cells, treat immune system disorders, treat metabolicor endocrine disorders, among other functions. For instance,Erythropoietin (EPO), a protein hormone can be utilized in treatingpatients with erythrocyte deficiency, which is a common cause of kidneycomplications. Furthermore adjuvant proteins, therapeutic antibodies areencompassed by therapeutic proteins and also hormone replacement therapywhich is e.g. used in the therapy of women in menopause. In more recentapproaches, somatic cells of a patient are used to reprogram them intopluripotent stem cells, which replace the disputed stem cell therapy.Also these proteins used for reprogramming of somatic cells or used fordifferentiating of stem cells are defined herein as therapeuticproteins. Furthermore, therapeutic proteins may be used for otherpurposes, e.g. wound healing, tissue regeneration, angiogenesis, etc.Furthermore, antigen-specific B cell receptors and fragments andvariants thereof are defined herein as therapeutic proteins.

Therefore therapeutic proteins can be used for various purposesincluding treatment of various diseases like e.g. infectious diseases,neoplasms (e.g. cancer or tumour diseases), diseases of the blood andblood-forming organs, endocrine, nutritional and metabolic diseases,diseases of the nervous system, diseases of the circulatory system,diseases of the respiratory system, diseases of the digestive system,diseases of the skin and subcutaneous tissue, diseases of themusculoskeletal system and connective tissue, and diseases of thegenitourinary system, independently if they are inherited or acquired.

In this context, particularly preferred therapeutic proteins, which canbe used inter alia in the treatment of metabolic or endocrine disorders,are selected from (in brackets the particular disease for which thetherapeutic protein is used in the treatment): Acid sphingomyelinase(Niemann-Pick disease), Adipotide (obesity), Agalsidase-beta (humangalactosidase A) (Fabry disease; prevents accumulation of lipids thatcould lead to renal and cardiovascular complications), Alglucosidase(Pompe disease (glycogen storage disease type II)), alpha-galactosidaseA (alpha-GAL A, Agalsidase alpha) (Fabry disease), alpha-glucosidase(Glycogen storage disease (GSD), Morbus Pompe), alpha-L-iduronidase(mucopolysaccharidoses (MPS), Hurler syndrome, Scheie syndrome),alpha-N-acetylglucosaminidase (Sanfilippo syndrome), Amphiregulin(cancer, metabolic disorder), Angiopoietin ((Ang1, Ang2, Ang3, Ang4,ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7) (angiogenesis,stabilize vessels), Betacellulin (metabolic disorder),Beta-glucuronidase (Sly syndrome), Bone morphogenetic protein BMPs(BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15)(regenerative effect, bone-related conditions, chronic kidney disease(CKD)), CLN6 protein (CLN6 disease—Atypical Late Infantile, Late Onsetvariant, Early Juvenile, Neuronal Ceroid Lipofuscinoses (NCL)),Epidermal growth factor (EGF) (wound healing, regulation of cell growth,proliferation, and differentiation), Epigen (metabolic disorder),Epiregulin (metabolic disorder), Fibroblast Growth Factor (FGF, FGF-1,FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11,FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20,FGF-21, FGF-22, FGF-23) (wound healing, angiogenesis, endocrinedisorders, tissue regeneration), Galsulphase (Mucopolysaccharidosis VI),Ghrelin (irritable bowel syndrome (IBS), obesity, Prader-Willi syndrome,type II diabetes mellitus), Glucocerebrosidase (Gaucher's disease),GM-CSF (regenerative effect, production of white blood cells, cancer),Heparin-binding EGF-like growth factor (HB-EGF) (wound healing, cardiachypertrophy and heart development and function), Hepatocyte growthfactor HGF (regenerative effect, wound healing), Hepcidin (ironmetabolism disorders, Beta-thalassemia), Human albumin (Decreasedproduction of albumin (hypoproteinaemia), increased loss of albumin(nephrotic syndrome), hypovolaemia, hyperbilirubinaemia), Idursulphase(Iduronate-2-sulphatase) (Mucopolysaccharidosis II (Hunter syndrome)),Integrins αVβ3, αV65 and α5β1 (Bind matrix macromolecules andproteinases, angiogenesis), luduronate sulfatase (Hunter syndrome),Laronidase (Hurler and Hurler-Scheie forms of mucopolysaccharidosis I),N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase, Arylsulfatase A(ARSA), Arylsulfatase B (ARSB)) (arylsulfatase B deficiency,Maroteaux-Lamy syndrome, mucopolysaccharidosis VI),N-acetylglucosamine-6-sulfatase (Sanfilippo syndrome), Nerve growthfactor (NGF, Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3(NT-3), and Neurotrophin 4/5 (NT-4/5) (regenerative effect,cardiovascular diseases, coronary atherosclerosis, obesity, type 2diabetes, metabolic syndrome, acute coronary syndromes, dementia,depression, schizophrenia, autism, Rett syndrome, anorexia nervosa,bulimia nervosa, wound healing, skin ulcers, corneal ulcers, Alzheimer'sdisease), Neuregulin (NRG1, NRG2, NRG3, NRG4) (metabolic disorder,schizophrenia), Neuropilin (NRP-1, NRP-2) (angiogenesis, axon guidance,cell survival, migration), Obestatin (irritable bowel syndrome (IBS),obesity, Prader-Willi syndrome, type II diabetes mellitus), PlateletDerived Growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D)(regenerative effect, wound healing, disorder in angiogenesis,Arteriosclerosis, Fibrosis, cancer), TGF beta receptors (endoglin,TGF-beta 1 receptor, TGF-beta 2 receptor, TGF-beta 3 receptor) (renalfibrosis, kidney disease, diabetes, ultimately end-stage renal disease(ESRD), angiogenesis), Thrombopoietin (THPO) (Megakaryocyte growth anddevelopment factor (MGDF)) (platelets disorders, platelets for donation,recovery of platelet counts after myelosuppressive chemotherapy),Transforming Growth factor (TGF (TGF-alpha, TGF-beta (TGFbeta1,TGFbeta2, and TGFbeta3))) (regenerative effect, wound healing, immunity,cancer, heart disease, diabetes, Marfan syndrome, Loeys-Dietz syndrome),VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF)(regenerative effect, angiogenesis, wound healing, cancer,permeability), Nesiritide (Acute decompensated congestive heartfailure), Trypsin (Decubitus ulcer, varicose ulcer, debridement ofeschar, dehiscent wound, sunburn, meconium ileus), adrenocorticotrophichormone (ACTH) (“Addison's disease, Small cell carcinoma,Adrenoleukodystrophy, Congenital adrenal hyperplasia, Cushing'ssyndrome, Nelson's syndrome, Infantile spasms), Atrial-natriureticpeptide (ANP) (endocrine disorders), Cholecystokinin (diverse), Gastrin(hypogastrinemia), Leptin (Diabetes, hypertriglyceridemia, obesity),Oxytocin (stimulate breastfeeding, non-progression of parturition),Somatostatin (symptomatic treatment of carcinoid syndrome, acutevariceal bleeding, and acromegaly, polycystic diseases of the liver andkidney, acromegaly and symptoms caused by neuroendocrine tumors),Vasopressin (antidiuretic hormone) (diabetes insipidus), Calcitonin(Postmenopausal osteoporosis, Hypercalcaemia, Paget's disease, Bonemetastases, Phantom limb pain, Spinal Stenosis), Exenatide (Type 2diabetes resistant to treatment with metformin and a sulphonylurea),Growth hormone (GH), somatotropin (Growth failure due to GH deficiencyor chronic renal insufficiency, Prader-Willi syndrome, Turner syndrome,AIDS wasting or cachexia with antiviral therapy), Insulin (Diabetesmellitus, diabetic ketoacidosis, hyperkalaemia), Insulin-like growthfactor 1 IGF-1 (Growth failure in children with GH gene deletion orsevere primary IGF1 deficiency, neurodegenerative disease,cardiovascular diseases, heart failure), Mecasermin rinfabate, IGF-1analog (Growth failure in children with GH gene deletion or severeprimary IGF1 deficiency, neurodegenerative disease, cardiovasculardiseases, heart failure), Mecasermin, IGF-1 analog (Growth failure inchildren with GH gene deletion or severe primary IGF1 deficiency,neurodegenerative disease, cardiovascular diseases, heart failure),Pegvisomant (Acromegaly), Pramlintide (Diabetes mellitus, in combinationwith insulin), Teriparatide (human parathyroid hormone residues 1-34)(Severe osteoporosis), Becaplermin (Debridement adjunct for diabeticulcers), Dibotermin-alpha (Bone morphogenetic protein 2) (Spinal fusionsurgery, bone injury repair), Histrelin acetate (gonadotropin releasinghormone; GnRH) (Precocious puberty), Octreotide (Acromegaly, symptomaticrelief of VIP-secreting adenoma and metastatic carcinoid tumours), andPalifermin (keratinocyte growth factor; KGF) (Severe oral mucositis inpatients undergoing chemotherapy, wound healing).

These and other proteins are understood to be therapeutic, as they aremeant to treat the subject by replacing its defective endogenousproduction of a functional protein in sufficient amounts. Accordingly,such therapeutic proteins are typically mammalian, in particular humanproteins.

For the treatment of blood disorders, diseases of the circulatorysystem, diseases of the respiratory system, cancer or tumour diseases,infectious diseases or immunedeficiencies following therapeutic proteinsmay be used: Alteplase (tissue plasminogen activator; tPA) (Pulmonaryembolism, myocardial infarction, acute ischaemic stroke, occlusion ofcentral venous access devices), Anistreplase (Thrombolysis),Antithrombin III (AT-Ill) (Hereditary AT-III deficiency,Thromboembolism), Bivalirudin (Reduce blood-clotting risk in coronaryangioplasty and heparin-induced thrombocytopaenia), Darbepoetin-alpha(Treatment of anaemia in patients with chronic renal insufficiency andchronic renal failure (+/−dialysis)), Drotrecogin-alpha (activatedprotein C) (Severe sepsis with a high risk of death), Erythropoietin,Epoetin-alpha, erythropoetin, erthropoyetin (Anaemia of chronic disease,myleodysplasia, anaemia due to renal failure or chemotherapy,preoperative preparation), Factor IX (Haemophilia B), Factor Vila(Haemorrhage in patients with haemophilia A or B and inhibitors tofactor VIII or factor IX), Factor VIII (Haemophilia A), Lepirudin(Heparin-induced thrombocytopaenia), Protein C concentrate (Venousthrombosis, Purpura fulminans), Reteplase (deletion mutein of tPA)(Management of acute myocardial infarction, improvement of ventricularfunction), Streptokinase (Acute evolving transmural myocardialinfarction, pulmonary embolism, deep vein thrombosis, arterialthrombosis or embolism, occlusion of arteriovenous cannula),Tenecteplase (Acute myocardial infarction), Urokinase (Pulmonaryembolism), Angiostatin (Cancer), Anti-CD22 immunotoxin (Relapsed CD33+acute myeloid leukaemia), Denileukin diftitox (Cutaneous T-cell lymphoma(CTCL)), Immunocyanin (bladder and prostate cancer), MPS(Metallopanstimulin) (Cancer), Aflibercept (Non-small cell lung cancer(NSCLC), metastatic colorectal cancer (mCRC), hormone-refractorymetastatic prostate cancer, wet macular degeneration), Endostatin(Cancer, inflammatory diseases like rheumatoid arthritis as well asCrohn's disease, diabetic retinopathy, psoriasis, and endometriosis),Collagenase (Debridement of chronic dermal ulcers and severely burnedareas, Dupuytren's contracture, Peyronie's disease), Humandeoxy-ribonuclease I, dornase (Cystic fibrosis; decreases respiratorytract infections in selected patients with FVC greater than 40% ofpredicted), Hyaluronidase (Used as an adjuvant to increase theabsorption and dispersion of injected drugs, particularly anaestheticsin ophthalmic surgery and certain imaging agents), Papain (Debridementof necrotic tissue or liquefication of slough in acute and chroniclesions, such as pressure ulcers, varicose and diabetic ulcers, burns,postoperative wounds, pilonidal cyst wounds, carbuncles, and otherwounds), L-Asparaginase (Acute lymphocytic leukaemia, which requiresexogenous asparagine for proliferation), Peg-asparaginase (Acutelymphocytic leukaemia, which requires exogenous asparagine forproliferation), Rasburicase (Paediatric patients with leukaemia,lymphoma, and solid tumours who are undergoing anticancer therapy thatmay cause tumour lysis syndrome), Human chorionic gonadotropin (HCG)(Assisted reproduction), Human follicle-stimulating hormone (FSH)(Assisted reproduction), Lutropin-alpha (Infertility with luteinizinghormone deficiency), Prolactin (Hypoprolactinemia, serum prolactindeficiency, ovarian dysfunction in women, anxiety, arteriogenic erectiledysfunction, premature ejaculation, oligozoospermia, asthenospermia,hypofunction of seminal vesicles, hypoandrogenism in men),alpha-1-Proteinase inhibitor (Congenital antitrypsin deficiency),Lactase (Gas, bloating, cramps and diarrhoea due to inability to digestlactose), Pancreatic enzymes (lipase, amylase, protease) (Cysticfibrosis, chronic pancreatitis, pancreatic insufficiency, post-BillrothII gastric bypass surgery, pancreatic duct obstruction, steatorrhoea,poor digestion, gas, bloating), Adenosine deaminase (pegademase bovine,PEG-ADA) (Severe combined immunodeficiency disease due to adenosinedeaminase deficiency), Abatacept (Rheumatoid arthritis (especially whenrefractory to TNFalpha inhibition)), Alefacept (Plaque Psoriasis),Anakinra (Rheumatoid arthritis), Etanercept (Rheumatoid arthritis,polyarticular-course juvenile rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, plaque psoriasis, ankylosing spondylitis),Interleukin-1 (IL-1) receptor antagonist, Anakinra (inflammation andcartilage degradation associated with rheumatoid arthritis), Thymulin(neurodegenerative diseases, rheumatism, anorexia nervosa), TNF-alphaantagonist (autoimmune disorders such as rheumatoid arthritis,ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitissuppurativa, refractory asthma), Enfuvirtide (HIV-1 infection), andThymosin al (Hepatitis B and C).

(in brackets is the particular disease for which the therapeutic proteinis used in the treatment)

In a further aspect, the present invention provides a vector comprisingan artificial nucleic acid molecule comprising an ORF and a 3′-UTR, the3′-UTR comprising at least one poly(A) sequence as defined herein,wherein the at least one poly(A) sequence comprises at least 70 adeninenucleotides. In a preferred embodiment, the artificial nucleic acidmolecule comprises a 3′-UTR that comprises at least two poly(A)sequences as defined herein.

The 3′-UTR and the ORF are as described above for the artificial nucleicacid molecule according to the present invention. The cloning site maybe any sequence that is suitable for introducing an open reading frameor a sequence comprising an open reading frame, such as one or morerestriction sites. Thus, the vector comprising a cloning site ispreferably suitable for inserting an open reading frame into the vector,preferably for inserting an open reading frame 5′ to the 3′-UTR.Preferably the cloning site or the ORF is located 5′ to the 3′-UTR,preferably in close proximity to the 5′-end of the 3′-UTR. For example,the cloning site or the ORF may be directly connected to the 5′-end ofthe 3′-UTR or they may be connected via a stretch of nucleotides, suchas by a stretch of 2, 4, 6, 8, 10, 20 etc. nucleotides as describedabove for the artificial nucleic acid molecule according to the presentinvention.

Preferably, the vector according to the present invention is suitablefor producing the artificial nucleic acid molecule according to thepresent invention, preferably for producing an artificial mRNA accordingto the present invention, for example, by optionally inserting an openreading frame or a sequence comprising an open reading frame into thevector and transcribing the vector. Thus, preferably, the vectorcomprises elements needed for transcription, such as a promoter, e.g. anRNA polymerase promoter. Preferably, the vector is suitable fortranscription using eukaryotic, prokaryotic, viral or phagetranscription systems, such as eukaryotic cells, prokaryotic cells, oreukaryotic, prokaryotic, viral or phage in vitro transcription systems.Thus, for example, the vector may comprise a promoter sequence, which isrecognized by a polymerase, such as by an RNA polymerase, e.g. by aeukaryotic, prokaryotic, viral, or phage RNA polymerase. In a preferredembodiment, the vector comprises a phage RNA polymerase promoter such asan SP6, T3 or T7, preferably a T7 promoter. Preferably, the vector issuitable for in vitro transcription using a phage based in vitrotranscription system, such as a T7 RNA polymerase based in vitrotranscription system.

In another preferred embodiment, the vector may be used directly forexpression of the encoded peptide or protein in cells or tissue. Forthis purpose, the vector comprises particular elements, which arenecessary for expression in those cells/tissue e.g. particular promotersequences, such as a CMV promoter.

The vector may further comprise a polyadenylation signal as describedabove for the artificial nucleic acid molecule according to the presentinvention.

The vector may be an RNA vector or a DNA vector. Preferably, the vectoris a DNA vector. The vector may be any vector known to the skilledperson, such as a viral vector or a plasmid vector. Preferably, thevector is a plasmid vector, preferably a DNA plasmid vector.

In a preferred embodiment of the invention, the present inventionprovides a vector comprising the artificial nucleic acid moleculeaccording to the invention.

Preferably, the vector is a circular molecule. Preferably, the vector isa double-stranded molecule, such as a double-stranded DNA molecule. Suchcircular, preferably double stranded DNA molecule may be usedconveniently as a storage form for the inventive artificial nucleic acidmolecule. Furthermore, it may be used for transfection of cells, forexample, cultured cells.

Also it may be used for in vitro transcription for obtaining anartificial RNA molecule according to the invention.

Preferably, the vector, preferably the circular vector, is linearizable,for example, by restriction enzyme digestion. In a preferred embodiment,the vector comprises a cleavage site, such as a restriction site,preferably a unique cleavage site, located immediately 3′ to the 3′-UTR,or located 3′ to the poly(A) sequence or—if present—to a polyadenylationsignal, or—if present—located 3′ to the poly(C) sequence, or—ifpresent—located 3′ to the histone stem-loop. Thus, preferably, theproduct obtained by linearizing the vector terminates at the 3′end withthe 3′-end of the 3′-UTR, or with the 3′-end of the poly(A) sequenceor—if present—polyadenylation signal, or—if present—with the 3′-end ofthe poly(C) sequence, or—if present—with the 3′-end of the histonestem-loop. In the embodiment, wherein the vector according to thepresent invention comprises the artificial nucleic acid moleculeaccording to the present invention, a restriction site, preferably aunique restriction site, is preferably located immediately 3′ to the3′-end of the artificial nucleic acid molecule. Preferably, arestriction site for linearization of the circular vector molecule islocated on the 3′ side of the 3′-UTR of the coding strand.

In a further aspect, the present invention relates to a cell comprisingthe artificial nucleic acid molecule according to the present inventionor the vector according to present invention. The cell may be any cell,such as a bacterial cell, insect cell, plant cell, vertebrate cell, e.g.a mammalian cell. Such cell may be, e.g., used for replication of thevector of the present invention, for example, in a bacterial cell.Furthermore, the cell may be used for transcribing the artificialnucleic acid molecule or the vector according to the present inventionand/or translating the open reading frame of the artificial nucleic acidmolecule or the vector according to the present invention. For example,the cell may be used for recombinant protein production.

The cells according to the present invention are, for example,obtainable by standard nucleic acid transfer methods, such as standardtransfection, transduction or transformation methods. For example, theartificial nucleic acid molecule or the vector according to the presentinvention may be transferred into the cell by electroporation,lipofection, e.g. based on cationic lipids and/or liposomes, calciumphosphate precipitation, nanoparticle based transfection, virus basedtransfection, or based on cationic polymers, such as DEAE-dextran orpolyethylenimine etc.

Preferably, the cell is a mammalian cell, such as a cell of humansubject, a domestic animal, a laboratory animal, such as a mouse or ratcell. Preferably, the cell is a human cell. The cell may be a cell of anestablished cell line, such as a CHO, BHK, 293T, COS-7, HELA, HEK, etc.or the cell may be a primary cell, such as a human dermal fibroblast(HDF) cell etc., preferably a cell isolated from an organism. In apreferred embodiment, the cell is an isolated cell of a mammaliansubject, preferably of a human subject. For example, the cell may be animmune cell, such as a dendritic cell, a cancer or tumor cell, or anysomatic cell etc., preferably of a mammalian subject, preferably of ahuman subject.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising the artificial nucleic acid molecule according tothe present invention, the vector according the present invention, orthe cell according to the present invention. The pharmaceuticalcomposition according to the invention may be used, e.g., as a vaccine,for example, for genetic vaccination. Thus, the ORF may, e.g., encode anantigen to be administered to a patient for vaccination. Thus, in apreferred embodiment, the pharmaceutical composition according to thepresent invention is a vaccine. Furthermore, the pharmaceuticalcomposition according to the present invention may be used, e.g., forgene therapy.

Preferably, the pharmaceutical composition further comprises one or morepharmaceutically acceptable vehicles, diluents and/or excipients and/orone or more adjuvants. In the context of the present invention, apharmaceutically acceptable vehicle typically includes a liquid ornon-liquid basis for the inventive pharmaceutical composition. In oneembodiment, the pharmaceutical composition is provided in liquid form.In this context, preferably, the vehicle is based on water, such aspyrogen-free water, isotonic saline or buffered (aqueous) solutions, e.gphosphate, citrate etc. buffered solutions. The buffer may behypertonic, isotonic or hypotonic with reference to the specificreference medium, i.e. the buffer may have a higher, identical or lowersalt content with reference to the specific reference medium, whereinpreferably such concentrations of the afore mentioned salts may be used,which do not lead to damage of mammalian cells due to osmosis or otherconcentration effects. Reference media are e.g. liquids occurring in “invivo” methods, such as blood, lymph, cytosolic liquids, or other bodyliquids, or e.g. liquids, which may be used as reference media in “invitro” methods, such as common buffers or liquids. Such common buffersor liquids are known to a skilled person. Ringer-Lactate solution isparticularly preferred as a liquid basis.

One or more compatible solid or liquid fillers or diluents orencapsulating compounds suitable for administration to a patient may beused as well for the inventive pharmaceutical composition. The term“compatible” as used herein preferably means that these components ofthe inventive pharmaceutical composition are capable of being mixed withthe inventive artificial nucleic acid, vector or cells as defined hereinin such a manner that no interaction occurs which would substantiallyreduce the pharmaceutical effectiveness of the inventive pharmaceuticalcomposition under typical use conditions.

The pharmaceutical composition according to the present invention mayoptionally further comprise one or more additional pharmaceuticallyactive components. A pharmaceutically active component in this contextis a compound that exhibits a therapeutic effect to heal, ameliorate orprevent a particular indication or disease. Such compounds include,without implying any limitation, peptides or proteins, nucleic acids,(therapeutically active) low molecular weight organic or inorganiccompounds (molecular weight less than 5000, preferably less than 1000),sugars, antigens or antibodies, therapeutic agents already known in theprior art, antigenic cells, antigenic cellular fragments, cellularfractions, cell wall components (e.g. polysaccharides), modified,attenuated or de-activated (e.g. chemically or by irradiation) pathogens(virus, bacteria etc.).

Furthermore, the inventive pharmaceutical composition may comprise acarrier for the artificial nucleic acid molecule or the vector. Such acarrier may be suitable for mediating dissolution in physiologicalacceptable liquids, transport and cellular uptake of the pharmaceuticalactive artificial nucleic acid molecule or the vector. Accordingly, sucha carrier may be a component, which may be suitable for depot anddelivery of an artificial nucleic acid molecule or vector according tothe invention. Such components may be, for example, cationic orpolycationic carriers or compounds, which may serve as transfection orcomplexation agent.

Particularly preferred transfection or complexation agents in thiscontext are cationic or polycationic compounds, including protamine,nucleoline, spermine or spermidine, or other cationic peptides orproteins, such as poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs), PpT620, proline-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, plsl, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, or histones.

Furthermore, such cationic or polycationic compounds or carriers may becationic or polycationic peptides or proteins, which preferably compriseor are additionally modified to comprise at least one —SH moiety.Preferably, a cationic or polycationic carrier is selected from cationicpeptides having the following sum formula (I):{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};  formula (I)

wherein l+m+n+o+x=3-100, and l, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may bepositioned at any place of the peptide. In this context, cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred.

Further, the cationic or polycationic peptide or protein, when definedaccording to formula {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}(formula (I)) as shown above and which comprise or are additionallymodified to comprise at least one —SH moeity, may be, without beingrestricted thereto, selected from subformula (Ia):{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)}  subformula(Ia)

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80 and 81-90, provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide. Further, the cationic orpolycationic peptide may be selected from subformula (Ib):Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂  subformula(Ib)

wherein empirical formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (III)) isas defined herein and forms a core of an amino acid sequence accordingto (semiempirical) formula (III) and wherein Cys₁ and Cys₂ are Cysteinesproximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x).

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene, cationic polymers,e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac42(2,3-dihexadecyloxypropyl-oxymethyloxy)ethylHrimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g polyethyleneglycole); etc.

According to another embodiment, the pharmaceutical compositionaccording to the invention may comprise an adjuvant in order to enhancethe immunostimulatory properties of the pharmaceutical composition. Inthis context, an adjuvant may be understood as any compound, which issuitable to support administration and delivery of the components suchas the artificial nucleic acid molecule or vector comprised in thepharmaceutical composition according to the invention. Furthermore, suchan adjuvant may, without being bound thereto, initiate or increase animmune response of the innate immune system, i.e. a non-specific immuneresponse. With other words, when administered, the pharmaceuticalcomposition according to the invention typically initiates an adaptiveimmune response directed to the antigen encoded by the artificialnucleic acid molecule. Additionally, the pharmaceutical compositionaccording to the invention may generate an (supportive) innate immuneresponse due to addition of an adjuvant as defined herein to thepharmaceutical composition according to the invention.

Such an adjuvant may be selected from any adjuvant known to a skilledperson and suitable for the present case, i.e. supporting the inductionof an immune response in a mammal. Preferably, the adjuvant may beselected from the group consisting of, without being limited thereto,TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminiumhydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucansfrom algae; algammulin; aluminium hydroxide gel (alum); highlyprotein-adsorbing aluminium hydroxide gel; low viscosity aluminiumhydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%),Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE™(propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DM PC(dimyristoylphosphatidylcholine); DM PG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP),ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline saltcomplex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferon-gamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA(polymethyl methacrylate); PODDS™ (proteinoid microspheres);polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylicacid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™(QS-21); Quil-A (Quil-A saponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5c]quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendaiproteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitantrioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85);squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin; microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, 1031, Imidazoquinolines, Ampligen, Ribi529, IMOxine,IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPIanchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusionprotein, cdiGMP; and adjuvants suitable as antagonists including CGRPneuropeptide.

Suitable adjuvants may also be selected from cationic or polycationiccompounds wherein the adjuvant is preferably prepared upon complexingthe artificial nucleic acid molecule or the vector of the pharmaceuticalcomposition with the cationic or polycationic compound. Association orcomplexing the artificial nucleic acid molecule or the vector of thepharmaceutical composition with cationic or polycationic compounds asdefined herein preferably provides adjuvant properties and confers astabilizing effect to the artificial nucleic acid molecule or the vectorof the pharmaceutical composition. Particularly such preferred, suchcationic or polycationic compounds are selected from cationic orpolycationic peptides or proteins, including protamine, nucleoline,spermin or spermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine,spermine, spermidine, or histones. Further preferred cationic orpolycationic compounds may include cationic polysaccharides, for examplechitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI),cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, Chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,Blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected of a cationic polymer as mentioned above) and of one ormore hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); etc.

Additionally, preferred cationic or polycationic proteins or peptides,which can be used as an adjuvant by complexing the artificial nucleicacid molecule or the vector, preferably an RNA, of the composition, maybe selected from following proteins or peptides having the followingtotal formula (I): (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x),wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred oligoarginines in this context are e.g. Arg₇,Arg₈, Arg₉, Arg₇, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc.

In a preferred embodiment, the artificial nucleic acid molecule,preferably an RNA molecule, is associated with or complexed with acationic or polycationic compound or a polymeric carrier, optionally ina weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1(w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), evenmore preferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)to about 2:1 (w/w) of RNA to cationic or polycationic compound and/orwith a polymeric carrier; or optionally in a nitrogen/phosphate ratio ofRNA to cationic or polycationic compound and/or polymeric carrier in therange of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1,and most preferably in a range of about 0.5-1 or 0.7-1, and even mostpreferably in a range of about 0.3-0.9 or 0.5-0.9.

The ratio of the artificial nucleic acid or the vector to the cationicor polycationic compound may be calculated on the basis of thenitrogen/phosphate ratio (N/P-ratio) of the entire nucleic acid complex.For example, 1 μg RNA typically contains about 3 nmol phosphateresidues, provided the RNA exhibits a statistical distribution of bases.Additionally, 1 μg peptide typically contains about x nmol nitrogenresidues, dependent on the molecular weight and the number of basicamino acids. When exemplarily calculated for (Arg)₉ (molecular weight1424 g/mol, 9 nitrogen atoms), 1 μg (Arg)₉ contains about 700 pmol(Arg)₉ and thus 700×9=6300 pmol basic amino acids=6.3 nmol nitrogenatoms. For a mass ratio of about 1:1 RNA/(Arg)₉ an N/P ratio of about 2can be calculated. When exemplarily calculated for protamine (molecularweight about 4250 g/mol, 21 nitrogen atoms, when protamine from salmonis used) with a mass ratio of about 2:1 with 2 μg RNA, 6 nmol phosphateare to be calulated for the RNA; 1 μg protamine contains about 235 pmolprotamine molecues and thus 235×21=4935 pmol basic nitrogen atoms=4.9nmol nitrogen atoms. For a mass ratio of about 2:1 RNA/protamine an N/Pratio of about 0.81 can be calculated. For a mass ratio of about 8:1RNA/protamine an N/P ratio of about 0.2 can be calculated. In thecontext of the present invention, an N/P-ratio is preferably in therange of about 0.1-10, preferably in a range of about 0.3-4 and mostpreferably in a range of about 0.5-2 or 0.7-2 regarding the ratio ofnucleic acid:peptide in the complex, and most preferably in the range ofabout 0.7-1.5.

Patent application WO2010/037539, the disclosure of which isincorporated herein by reference, describes an immunostimulatorycomposition and methods for the preparation of an immunostimulatorycomposition. Accordingly, in a preferred embodiment of the invention,the composition is obtained in two separate steps in order to obtainboth, an efficient immunostimulatory effect and efficient translation ofthe artificial nucleic acid molecule according to the invention.Therein, a so called “adjuvant component” is prepared by complexing—in afirst step—the artificial nucleic acid molecule or vector, preferably anRNA, of the adjuvant component with a cationic or polycationic compoundin a specific ratio to form a stable complex. In this context, it isimportant, that no free cationic or polycationic compound or only aneglibly small amount remains in the adjuvant component after complexingthe nucleic acid. Accordingly, the ratio of the nucleic acid and thecationic or polycationic compound in the adjuvant component is typicallyselected in a range that the nucleic acid is entirely complexed and nofree cationic or polycationic compound or only a neclectably smallamount remains in the composition. Preferably the ratio of the adjuvantcomponent, i.e. the ratio of the nucleic acid to the cationic orpolycationic compound is selected from a range of about 6:1 (w/w) toabout 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1(w/w), even more preferably of about 4:1 (w/w) to about 1:1 (w/w) or ofabout 3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about3:1 (w/w) to about 2:1 (w/w).

According to a preferred embodiment, the artificial nucleic acidmolecule or vector, preferably an RNA molecule, according to theinvention is added in a second step to the complexed nucleic acidmolecule, preferably an RNA, of the adjuvant component in order to formthe (immunostimulatory) composition of the invention. Therein, theartificial acid molecule or vector, preferably an RNA, of the inventionis added as free nucleic acid, i.e. nucleic acid, which is not complexedby other compounds. Prior to addition, the free artificial nucleic acidmolecule or vector is not complexed and will preferably not undergo anydetectable or significant complexation reaction upon the addition of theadjuvant component.

Suitable adjuvants may furthermore be selected from nucleic acids havingthe formula (II): G_(l)X_(m)G_(n), wherein: G is guanosine, uracil or ananalogue of guanosine or uracil; X is guanosine, uracil, adenosine,thymidine, cytosine or an analogue of the above-mentioned nucleotides; lis an integer from 1 to 40, wherein when l=1 G is guanosine or ananalogue thereof, when l>1 at least 50% of the nucleotides are guanosineor an analogue thereof; m is an integer and is at least 3; wherein whenm=3 X is uracil or an analogue thereof, when m>3 at least 3 successiveuracils or analogues of uracil occur; n is an integer from 1 to 40,wherein when n=1 G is guanosine or an analogue thereof, when n>1 atleast 50% of the nucleotides are guanosine or an analogue thereof.

Other suitable adjuvants may furthermore be selected from nucleic acidshaving the formula (III): C_(l)X_(m)C_(n), wherein: C is cytosine,uracil or an analogue of cytosine or uracil; X is guanosine, uracil,adenosine, thymidine, cytosine or an analogue of the above-mentionednucleotides; l is an integer from 1 to 40, wherein when l=1 C iscytosine or an analogue thereof, when l>1 at least 50% of thenucleotides are cytosine or an analogue thereof; m is an integer and isat least 3; wherein when m=3 X is uracil or an analogue thereof, whenm>3 at least 3 successive uracils or analogues of uracil occur; n is aninteger from 1 to 40, wherein when n=1 C is cytosine or an analoguethereof, when n>1 at least 50% of the nucleotides are cytosine or ananalogue thereof.

The pharmaceutical composition according to the present inventionpreferably comprises a “safe and effective amount” of the components ofthe pharmaceutical composition, particularly of the inventive artificialnucleic acid molecule, the vector and/or the cells as defined herein. Asused herein, a “safe and effective amount” means an amount sufficient tosignificantly induce a positive modification of a disease or disorder asdefined herein. At the same time, however, a “safe and effective amount”preferably avoids serious side-effects and permits a sensiblerelationship between advantage and risk. The determination of theselimits typically lies within the scope of sensible medical judgment.

In a further aspect, the present invention provides the artificialnucleic acid molecule according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention, or the pharmaceutical composition according to the presentinvention for use as a medicament, for example, as vaccine (in geneticvaccination) or in gene therapy.

The artificial nucleic acid molecule according to the present invention,the vector according to the present invention, the cell according to thepresent invention, or the pharmaceutical composition according to thepresent invention are particularly suitable for any medical application,which makes use of the therapeutic action or effect of peptides,polypeptides or proteins, or where supplementation of a particularpeptide or protein is needed. Thus, the present invention provides theartificial nucleic acid molecule according to the present invention, thevector according to the present invention, the cell according to thepresent invention, or the pharmaceutical composition according to thepresent invention for use in the treatment or prevention of diseases ordisorders amenable to treatment by the therapeutic action or effect ofpeptides, polypeptides or proteins or amenable to treatment bysupplementation of a particular peptide, polypeptide or protein. Forexample, the artificial nucleic acid molecule according to the presentinvention, the vector according to the present invention, the cellaccording to the present invention, or the pharmaceutical compositionaccording to the present invention may be used for the treatment orprevention of genetic diseases, autoimmune diseases, cancerous ortumour-related diseases, infectious diseases, chronic diseases or thelike, e.g., by genetic vaccination or gene therapy.

In particular, such therapeutic treatments, which benefit from a stableand prolonged presence of therapeutic peptides, polypeptides or proteinsin a subject to be treated, are especially suitable as medicalapplication in the context of the present invention, since the inventiveartificial nucleic acid molecule provides for a stable, increased and/orprolonged expression of the peptide or protein encoded by the inventiveartificial nucleic acid molecule or vector. Thus, a particularlysuitable medical application for the artificial nucleic acid moleculeaccording to the present invention, the vector according to the presentinvention, the cell according to the present invention, or thepharmaceutical composition according to the present invention isvaccination. Thus, the present invention provides the artificial nucleicacid molecule according to the present invention, the vector accordingto the present invention, the cell according to the present invention,or the pharmaceutical composition according to the present invention forvaccination of a subject, preferably a mammalian subject, morepreferably a human subject. Preferred vaccination treatments arevaccination against infectious diseases, such as bacterial, protozoal orviral infections, and anti-tumour-vaccination. Such vaccinationtreatments may be prophylactic or therapeutic.

Depending on the disease to be treated or prevented, the ORF may beselected. For example, the open reading frame may encode a protein thathas to be supplied to a patient suffering from total lack or at leastpartial loss of function of a protein, such as a patient suffering froma genetic disease. Additionally the open reading frame may be chosenfrom an ORF encoding a peptide or protein, which beneficially influencesa disease or the condition of a subject. Furthermore, the open readingframe may encode a peptide or protein, which results in thedownregulation of a pathological overproduction of a natural peptide orprotein or elimination of cells expressing pathologically a protein orpeptide. Such lack, loss of function or overproduction may, e.g., occurin the context of tumour and neoplasia, autoimmune diseases, allergies,infections, chronic diseases or the like. Furthermore, the open readingframe may encode an antigen or immunogen, e.g. an epitope of a pathogenor a tumour antigen. Thus, in preferred embodiments, the artificialnucleic acid molecule or the vector according to the present inventioncomprises an ORF encoding an amino acid sequence comprising orconsisting of an antigen or immunogen, e.g. an epitope of a pathogen ora tumour-associated antigen, and a 3′-UTR as described above.

In the context of medical applications, particularly in the context ofvaccination, it is preferred that the artificial nucleic acid moleculeaccording to the present invention is RNA, preferably mRNA, since DNAharbours the risk of eliciting an anti-DNA immune response and tends toinsert into genomic DNA. However, in some embodiments, for example, if aviral delivery vehicle, such as an adenoviral delivery vehicle is usedfor delivery of the artificial nucleic acid molecule or the vectoraccording to the present invention, e.g., in the context of genetherapeutic treatments, it may be desirable that the artificial nucleicacid molecule or the vector is a DNA molecule.

The artificial nucleic acid molecule according to the present invention,the vector according to the present invention, the cell according to thepresent invention, or the pharmaceutical composition according to thepresent invention are administered orally, parenterally, by inhalationspray, topically, rectally, nasally, buccally, vaginally, via animplanted reservoir or via jet injection. The term parenteral as usedherein includes subcutaneous, intravenous, intramuscular,intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic,intralesional, intracranial, transdermal, intradermal, intrapulmonal,intraperitoneal, intracardial, intraarterial, and sublingual injectionor infusion techniques. In a preferred embodiment, the artificialnucleic acid molecule according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention, or the pharmaceutical composition according to the presentinvention is administered intramuscularly, preferably via conventionalneedle injection or via needle-free injection (e.g. jet injection).

Preferably, the artificial nucleic acid molecule according to thepresent invention, the vector according to the present invention, thecell according to the present invention, or the pharmaceuticalcomposition according to the present invention is administeredparenterally, e.g. by parenteral injection, more preferably bysubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional,intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal,intracardial, intraarterial, sublingual injection or via infusiontechniques. Particularly preferred is intramuscular injection. Sterileinjectable forms of the inventive pharmaceutical composition may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. Preferably, the solutions orsuspensions are administered via conventional needle injection or vianeedle-free injection (e.g. jet injection).

The artificial nucleic acid molecule according to the present invention,the vector according to the present invention, the cell according to thepresent invention, or the pharmaceutical composition according to thepresent invention may also be administered orally in any orallyacceptable dosage form including, but not limited to, capsules, tablets,aqueous suspensions or solutions.

The artificial nucleic acid molecule according to the present invention,the vector according to the present invention, the cell according to thepresent invention, or the pharmaceutical composition according to thepresent invention may also be administered topically, especially whenthe target of treatment includes areas or organs readily accessible bytopical application, e.g. including diseases of the skin or of any otheraccessible epithelial tissue. Suitable topical formulations are readilyprepared for each of these areas or organs. For topical applications,the artificial nucleic acid molecule according to the present invention,the vector according to the present invention, the cell according to thepresent invention, or the pharmaceutical composition according to thepresent invention may be formulated in a suitable ointment suspended ordissolved in one or more carriers.

In one embodiment, the use as a medicament comprises the step oftransfection of mammalian cells, preferably in vitro or ex vivotransfection of mammalian cells, more preferably in vitro transfectionof isolated cells of a subject to be treated by the medicament. If theuse comprises the in vitro transfection of isolated cells, the use as amedicament may further comprise the readministration of the transfectedcells to the patient. The use of the inventive artificial nucleic acidmolecules or the vector as a medicament may further comprise the step ofselection of successfully transfected isolated cells. Thus, it may bebeneficial if the vector further comprises a selection marker. Also, theuse as a medicament may comprise in vitro transfection of isolated cellsand purification of an expression-product, i.e. the encoded peptide orprotein from these cells. This purified peptide or protein maysubsequently be administered to a subject in need thereof.

The present invention also provides a method for treating or preventinga disease or disorder as described above comprising administering theartificial nucleic acid molecule according to the present invention, thevector according to the present invention, the cell according to thepresent invention, or the pharmaceutical composition according to thepresent invention to a subject in need thereof. Therein, the artificialnucleic acid molecule, the vector, the cell or the pharmaceuticalcomposition are administered by any route as described herein.Particularly preferred, in this context, is the intramuscularadministration of the artificial nucleic acid molecule, the vector, thecell or the pharmaceutical composition.

According to a preferred embodiment, the inventive artificial nucleicacid molecule, the inventive vector, the inventive cell, the inventivevaccine or the inventive pharmaceutical composition is administered tosubject intramuscularly. Advantageously, intramuscular administration ofthe inventive nucleic acid molecule, vector, vaccine or compositionresults in an increased expression of the peptide or protein encoded bythe at least one open reading frame of the artificial nucleic acidmolecule as defined herein. In a particularly preferred embodiment, theexpression of the artificial nucleic acid molecule as defined herein,preferably of the artificial nucleic acid molecule comprising a 3′-UTRcomprising at least one, more preferably at least two, poly(A) sequencesas defined herein, is enhanced when administered intramuscularly. Mostpreferably, the intramuscular administration of the inventive artificialnucleic acid molecule, preferably of the inventive artificial nucleicacid molecule comprising at least two poly(A) sequences, results inincreased expression and in an improved immune response against anantigen, preferably a pathogenic antigen as defined herein, morepreferably an antigen associated with an infectious disease.

Furthermore, the present invention provides a method for treating orpreventing a disease or disorder comprising transfection of a cell withan artificial nucleic acid molecule according to the present inventionor with the vector according to the present invention. Said transfectionmay be performed in vitro, ex vivo or in vivo. In a preferredembodiment, transfection of a cell is performed in vitro and thetransfected cell is administered to a subject in need thereof,preferably to a human patient. Preferably, the cell, which is to betransfected in vitro, is an isolated cell of the subject, preferably ofthe human patient. Thus, the present invention provides a method oftreatment comprising the steps of isolating a cell from a subject,preferably from a human patient, transfecting the isolated cell with theartificial nucleic acid according to the present invention or the vectoraccording to the present invention, and administering the transfectedcell to the subject, preferably the human patient.

The method of treating or preventing a disorder according to the presentinvention is preferably a vaccination method or a gene therapy method asdescribed above.

As described above, the inventive 3′-UTR is capable of increasing theprotein production from an artificial nucleic acid molecule. Thus, in afurther aspect, the present invention relates to a method for increasingprotein production from an artificial nucleic acid molecule, preferablyfrom an mRNA molecule or a vector, the method comprising the step ofassociating the nucleic acid molecule, preferably the mRNA molecule orthe vector, with a 3′-untranslated region (3′-UTR), wherein the 3′-UTRcomprises at least one poly(A) sequence, wherein the at least onepoly(A) sequence comprises at least 70 adenosine residues, or apolyadenylation signal. In a preferred embodiment, the method comprisesassociating the nucleic acid molecule, preferably the mRNA or thevector, with a 3′-UTR comprising at least two poly(A) sequences asdescribed herein.

The term “associating the artificial nucleic acid molecule or the vectorwith a 3′-UTR” in the context of the present invention preferably meansfunctionally associating or functionally combining the artificialnucleic acid molecule or the vector with the 3′-UTR. This means that theartificial nucleic acid molecule or the vector and the 3′-UTR,preferably the 3′-UTR as described above, are associated or coupled suchthat the function of the 3′-UTR, e.g., the protein production increasingfunction, is exerted. Typically, this means that the 3′-UTR isintegrated into the artificial nucleic acid molecule or the vector,preferably the mRNA molecule, 3′ to an open reading frame, preferablyimmediately 3′ to an open reading frame, preferably between the openreading frame and a polyadenylation signal. Preferably, the 3′-UTR isintegrated into the artificial nucleic acid molecule or the vector,preferably the mRNA, as 3′-UTR, i.e. such that the 3′-UTR is the 3′-UTRof the artificial nucleic acid molecule or the vector, preferably themRNA, i.e., such that it extends from the 3′-side of the open readingframe to the 5′-terminus of the molecule or to the 5′-side of a poly(A)sequence or a polyadenylation signal, optionally connected via a shortlinker, such as a sequence comprising or consisting of one or morerestriction sites. Thus, preferably, the term “associating theartificial nucleic acid molecule or the vector with a 3′-UTR” meansfunctionally associating the 3′-UTR with an open reading frame locatedwithin the artificial nucleic acid molecule or the vector, preferablywithin the mRNA molecule. The 3′-UTR and the ORF are as described abovefor the artificial nucleic acid molecule according to the presentinvention, for example, preferably the ORF and the 3′-UTR areheterologous, e.g. derived from different genes, as described above.

In a preferred embodiment of the inventive method for increasing proteinproduction from an artificial nucleic acid molecule, preferably from anmRNA molecule or a vector, the at least one poly(A) sequence is producedby a chemical or enzymatic polyadenylation reaction. Preferably, abacterial enzyme, such as E. coli poly(A) polymerase, is employedtherein. In a preferred embodiment, the length of the at least onepoly(A) sequence is—amongst other parameters—regulated by the durationof the polyadenylation reaction, i.e. longer incubation of a nucleicacid with a suitable enzyme typically leads to a longer poly(A)sequence. Preferably, the nucleic acid molecule is incubated with asuitable enzyme for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85 or 90 minutes, preferably at a temperaturesuitable for the respective enzyme, e.g. 37° C. More preferably, thepolyadenylation reaction lasts from about 10 to about 120 minutes,preferably from about 15, 20, 25 or 30 to about 90 minutes, morepreferably from about 30 to about 60 minutes. In order to obtain apopulation of nucleic acid molecules, which share approximately the samedegree of polyadenylation, i.e. which have approximately the same numberof adenylates attached to their 3′-ends, the skilled person can choosefrom standard separation techniques (e.g. based on molecular weight orcharge), such as chromatographic methods, that are well-known in the artand that are typically employed after polyadenylation in order toseparate or purify the reaction products. A population of artificialnucleic acid molecules is preferably used according to the invention,which is more or less homogenous with respect to the length of the3′-terminal poly(A) sequence. Preferably, a population of artificialnucleic acid molecules is used, wherein at least 80%, more preferably atleast 85%, 90%, 95% or 98% of the molecules are characterized by thesame length of the 3′-terminal poly(A) sequence. In this context, ‘thesame length’ refers to a situation, where the number of adenylates inthe 3′-terminal poly(A) sequence varies from a given value (such as 160adenylates, 380 adenylates, 430 adenylates, 1000 adenylates, etc.) bynot more than 10%, more preferably not more than 9%, 8%, 7%, 6%, 5%, 4%,3%, 2% or not more than 1%.

In a further aspect, the present invention provides the use of a 3′-UTRfor increasing protein production from an artificial nucleic acidmolecule as described herein, preferably from an mRNA molecule or avector, wherein the 3′-UTR comprises at least one poly(A) sequence,wherein the at least one poly(A) sequence comprises at least 70adenosine residues, or a polyadenylation signal. In a preferredembodiment of the use according to the invention, a 3′-UTR comprising atleast two poly(A) sequences, preferably as described herein, is used forincreasing protein production from a nucleic acid molecule, preferablyfrom an artificial nucleic acid molecule as described herein.

The compounds and ingredients of the inventive pharmaceuticalcomposition may also be manufactured and traded separately of eachother. Thus, the invention relates further to a kit or kit of partscomprising an artificial nucleic acid molecule according to theinvention, a vector according to the invention, a cell according to theinvention, and/or a pharmaceutical composition according to theinvention. Preferably, such kit or kits of parts may, additionally,comprise instructions for use, cells for transfection, an adjuvant, ameans for administration of the pharmaceutical composition, apharmaceutically acceptable carrier and/or a pharmaceutically acceptablesolution for dissolution or dilution of the artificial nucleic acidmolecule, the vector, the cells or the pharmaceutical composition.

BRIEF DESCRIPTION OF THE FIGURES

The figures shown in the following are merely illustrative and shalldescribe the present invention in a further way. These figures shall notbe construed to limit the present invention thereto.

FIG. 1: DNA sequence (SEQ ID NO: 13) encoding the mRNA sequence, whichhas been used in the experiments and which comprises the sequencesencoding the following elements:

-   -   rpl32-PpLuc(GC)-albumin7-A64-C30-histone stem-loop.    -   Within the DNA sequence, the sequence elements corresponding to        the following elements in the mRNA are highlighted: PpLuc(GC)        (ORF) in italics, rpl32 (5′-UTR) underlined and albumin7        (3′-UTR) underlined.

FIGS. 2A-C: Polyadenylation of the mRNA sequence corresponding to SEQ IDNO: 13: A. Ca. 160 adenylates were added to one lot of mRNA (Lot 1). Ca.380 adenylates were added to a different lot of mRNA (Lot 2). mRNAcorresponding to SEQ ID NO: 13 was loaded onto the left lane, therespective adenylated mRNA was loaded onto the right line. A molecularsize marker was loaded on the outermost lanes for size comparison (thenumbers in FIG. 2A indicate the number of nucleotides comprised in themarker molecules; the same marker was used in FIG. 2B and FIG. 2C). B.Ca. 430 adenylates were added to the mRNA corresponding to SEQ ID NO:13. C. Ca. 1000 adenylates were added to the mRNA corresponding to SEQID NO: 13.

FIG. 3: Protein expression from polyadenylated mRNA in cultured cells:

-   -   mRNA corresponding to SEQ ID NO: 13, to which ca. 160 (mRNA        lot 1) or ca. 380 (mRNA lot 2) adenylates have been added by        polyadenylation, was transfected into human dermal fibroblasts        (HDF) and luciferase levels were measured at the indicated time        points. FIG. 3 shows the results as mean RLU (relative light        units)+/−SEM (standard error) for triplicate transfections.

FIG. 4: Protein expression from polyadenylated mRNA after intramuscularinjection into mice:

-   -   2 μg of mRNA corresponding to SEQ ID NO: 13, to which ca. 380        adenylates have been added by polyadenylation, were        intramuscularly injected into mice. FIG. 4 shows the results as        the median of up to 10 replicates.

FIG. 5: Protein expression from polyadenylated mRNA after intramuscularinjection into mice:

-   -   10 μg of mRNA corresponding to SEQ ID NO: 13, to which ca. 430        adenylates have been added by polyadenylation, were        intramuscularly injected into mice. FIG. 5 shows the results as        the median of up to 10 replicates.

FIG. 6: Protein expression from polyadenylated mRNA after intramuscularinjection into mice:

-   -   1 μg of mRNA corresponding to SEQ ID NO: 13, to which ca. 1000        adenylates have been added by polyadenylation, was        intramuscularly injected into mice. FIG. 6 shows the results as        the median of up to 10 replicates.

FIG. 7: Induction of HA neutralizing antibodies by polyadenylated mRNAafter intramuscular injection into mice:

FIG. 8: DNA sequence (SEQ ID NO: 14) encoding the mRNA sequence, whichhas been used in the experiments and which comprises the sequencesencoding the following elements:

-   -   32L4-H1N1 (Netherlands2009)-HA(GC)-albumin7-A64-N5-C30-histoneSL

FIG. 9: Induction of virus neutralizing titers by polyadenylated mRNAafter intramuscular injection into mice.

FIG. 10: DNA sequence (SEQ ID NO: 15) encoding the mRNA sequence, whichhas been used in the experiments and which comprises the sequencesencoding the following elements:

-   -   32L4-RAVG(GC)-albumin7-A64-N5-C30-histoneSL

EXAMPLES

The Examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These Examples shallnot be construed to limit the present invention thereto.

1. Preparation of DNA-Templates

A vector for in vitro transcription was constructed containing a T7promoter and a GC-enriched sequence encoding Photinus pyralis luciferase(PpLuc(GC)). The 5′ untranslated region (5′-UTR) of ribosomal proteinLarge 32 was inserted 5′ of PpLuc(GC). A 3′-UTR derived from humanalbumin (albumin7) was inserted 3′ of PpLuc(GC). Furthermore, an A64poly(A) sequence, followed by C30 and a histone stem-loop sequence, wasinserted 3′ of albumin7. The histone stem-loop sequence was followed bya restriction site used for linearization of the vector prior to invitro transcription. mRNA obtained from this vector accordingly by invitro transcription is designated as“rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL”.

In summary, a vector was generated that comprises the sequence, whichencodes the mRNA, which was used in further experiments. The DNAsequence (SEQ ID NO: 13) encoding said mRNA is shown in FIG. 1. The mRNAcorresponding to said DNA sequence is characterized by the followingelements:

rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL

Therein, the following abbreviations are used:

-   -   PpLuc (GC): GC-enriched mRNA sequence encoding Photinus pyralis        luciferase    -   rpl32: 5′-UTR of human ribosomal protein Large 32 lacking the 5′        terminal oligopyrimidine tract    -   albumin7: 3′-UTR of human albumin with three single point        mutations introduced to remove a T7 termination signal as well        as a HindIII and a XbaI restriction site    -   A64: poly(A)-sequence with 64 adenylates    -   C30: poly(C)-sequence with 30 cytidylates    -   histoneSL: histone stem-loop sequence according to SEQ ID NO:        11.

Further constructs used in the experiments:

32L4-H1N1 (Netherlands2009)-HA(GC)-albumin7-A64-N5-C30-histoneSL (SEQ IDNO: 14)

32L4-RAV-G(GC)-albumin7-A64-N5-C30-histoneSL (SEQ ID NO: 15)

The templates were prepared as described forrpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL.

2. In Vitro Transcription

The DNA template prepared in Example 1 was linearized and transcribed invitro using T7 polymerase. The DNA template was then digested by DNasetreatment. mRNA transcripts contained a 5′-cap structure obtained byadding an excess of N7-methyl-guanosine-5′-triphosphate-5′-guanosine tothe transcription reaction. mRNA thus obtained was purified andresuspended in water.

3. Enzymatic Adenylation

RNA was reacted with E. coli poly(A) polymerase (Cellscript) using 1 mMATP at 37° C. for 30 or 60 min. Immediately afterwards, RNA was purifiedusing a spin column (RNeasy mini column, Quiagen). RNA was run on a gelto assess RNA extension.

For vaccination experiments the mRNA was optionally complexed withprotamine. mRNA complexation consisted of a mixture of 50% naked mRNAand 50% mRNA complexed with protamine at a weight ratio of 2:1. First,mRNA was complexed with protamine by addition of protamine-Ringer'slactate solution to mRNA. After incubation for 10 minutes, when thecomplexes were stably generated, naked mRNA was added, and the finalconcentration of the vaccine was adjusted with Ringer's lactatesolution. The obtained formulated mRNA vaccine was used for in vivoexperiments.

4. Protein Expression by mRNA Lipofection

Human dermal fibroblasts (HDF) were seeded in 96-well plates three daysbefore transfection at a density of 10⁴ cells per well. Immediatelybefore lipofection, cells were washed in Opti-MEM. Cells were lipofectedwith 25 ng of PpLuc-encoding mRNA per well complexed withLipofectamine2000. mRNA encoding Renilla reniformis luciferase (RrLuc)was transfected together with PpLuc mRNA to control for transfectionefficiency (2.5 ng of RrLuc mRNA per well). 90 minutes after initiationof the transfection, Opti-MEM was exchanged for medium. 6, 24, 48, and72 hours after transfection, medium was aspirated and cells were lysedin 100 μl of lysis buffer (Passive Lysis Buffer, Promega). Lysates werestored at −80° C. until luciferase activity was measured.

5. Luminescence Measurement in Cell Lysate

Luciferase activity was measured as relative light units (RLU) in aHidex Chameleon plate reader. PpLuc activity was measured at 2 secondsmeasuring time using 20 μl of lysate and 50 μl of luciferin buffer(Beetle-Juice, PJK GmbH). RrLuc activity was measured at 2 secondsmeasuring time using 20 μl of lysate and 50 μl of coelenterazin buffer(Renilla-Juice, PJK GmbH).

6. Protein Expression by Intramuscular mRNA Injection

Mice were anaesthetized by intraperitoneal injection of a Ketavet andRompun mixture. After shaving the lower leg of the animal, 2 μg ofPpLuc-encoding mRNA in 20 μl of Ringer's lactate (80%) were injectedintramuscularly (M. tibialis or M. gastrocnemius).

7. In Vivo Luminescence Imaging

Mice were anaesthetized by intraperitoneal injection of a Ketavet andRompun mixture. 150 μl of Luciferin solution (20 g/l) were injectedintraperitoneally. 10 minutes after Luciferin injection, luminescencewas recorded on an IVIS Lumina II Imaging System.

Results

8.1 Additional Polyadenylation of the Artificial mRNA Increases ProteinExpression from the Artificial mRNA In Vitro

To investigate the effect of additional polyadenylation of theartificial mRNA on protein expression from the mRNA, the artificial mRNAwas synthesized by in vitro transcription(rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL). Part of one lot of mRNAwas enzymatically adenylated to add a poly(A) tail of ca. 160 adenylates(Lot 1). Part of a different lot of mRNA was enzymatically adenylated toadd a poly(A) tail of ca. 380 adenylates (Lot 2) (see FIG. 2).

Luciferase-encoding mRNAs were transfected into human dermal fibroblasts(HDF) in triplicate. Luciferase levels were measured at 6, 24, 48, and72 hours after transfection. From these data, total protein expressedfrom 0 to 72 hours was calculated as the area under the curve (AUC) (seefollowing Table 1 and FIG. 3).

TABLE 1 Luciferase activity measured in human dermal fibroblasts (HDF)RLU at RLU at RLU at RLU at Poly(A) tail 6 hours 24 hours 48 hours 72hours AUC Lot 1 incl. A64 37252 59085 29825 14612 2579000 Lot 1 incl.A64 81043 246102 89506 41308 8784000 plus ca. A160 (A224) Lot 2 incl.A64 47959 61053 23001 14053 2578000 Lot 2 incl. A64 69780 188560 6926944478 6993000 plus ca. A380 (A444)

Total Luciferase expression was identical from both mRNA lots containingan in vitro transcribed A64 sequence. The addition of ca. 160 adenylatesto the 3′ end of the mRNA (resulting in a 3′-UTR comprising ca. 224adenylates that are comprised in a poly(A) sequence) increasedluciferase expression by factor 3.4. Addition of ca. 380 adenylates tothe 3′ end of the mRNA (resulting in a 3′-UTR comprising ca. 444adenyates that are comprised in a poly(A) sequence) increased luciferaseexpression only to a similar extent, by factor 2.7.

Thus, addition of (further) adenylates to the 3′ end of the mRNAmarkedly increases the in vitro expression of the protein encoded by themRNA. In particular, a 3′-UTR comprising more than 64 adenylates thatare comprised in a poly(A) sequence markedly increases proteinexpression in vitro.

8.2 Additional Polyadenylation of the Artificial mRNA Strongly IncreasesProtein Expression from the Artificial mRNA after IntramuscularInjection

To investigate the effect of additional polyadenylation of theartificial mRNA on protein expression from the intramuscularly injectedmRNA, the artificial mRNA was synthesized by in vitro transcription(rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL). Part of this mRNA wasenzymatically adenylated to add a poly(A) tail of ca. 380 adenylates.

2 μg of luciferase-encoding mRNAs were injected intramuscularly (M.tibialis or M. gastrocnemius) in BALB/c mice (10 replicates per group).In vivo luminescence was recorded the following days (see FIG. 4). Fromthese data, total protein expressed from 0 to 15 days was calculated asthe area under the curve. Luciferase was clearly expressed fromintramuscularly injected mRNA containing an A64 sequence. Strikingly,however, additional polyadenylation of the artificial mRNA with 380adenylates increased luciferase expression very strongly, raising totalluciferase expressed eightfold. The magnitude of the rise in expressiondue to the (additional) poly(A) tail was unanticipated considering themuch smaller effect observed in cultured cells. Thus, the addition of(further) adenylates comprised in a poly(A) sequence increases proteinexpression from intramuscularly injected mRNA very strongly to anunanticipated extent. In parallel, the effect of the additionalpolyadenylation of the artificial mRNA with about 430 adenylates (seeFIG. 5) and about 1000 adenylates (see FIG. 6), respectively, wastested. While the both of the mRNAs that were polyadenylated with about430 adenylates and about 1000 adenylates, respectively, led to increasedprotein expression as compared to non-polyadenlyated mRNA, no furtherincrease was observed with respect to the artificial mRNA polyadenylatedwith 380 adenylates.

9. Vaccination with mRNA Encoding HA:

Balb/c mice were vaccinated 2 times (d0 and d21) into both M. tibialis.8 mice were vaccinated with 40 μg R2564 (naked HA mRNA), 8 animals werevaccinated with 40 μg polyadenylated R2564 (SEQ ID NO: 14; naked,polyadenylated HA), 8 animals were vaccinated with 40 μg R2630(formulated HA mRNA) and 8 animals were vaccinated with 40 μg firstpolyadenylated and then formulated R2564. 8 mice injected with RiLaserved as controls. Blood was collected on d35.

9.1. Hemagglutination Inhibition Assay (HI)

In a 96-well plate, the obtained sera were mixed with HA H1N1 antigen(A/California/07/2009 (H1N1); NIBSC) and red blood cells (4%erythrocytes; Lohmann Tierzucht). In the presence of HA neutralizingantibodies, an inhibition of hemagglutination of erythrocytes can beobserved. The lowest level of titered serum that resulted in a visibleinhibition of hemagglutination was the assay result.

Results:

The results show that higher HI titers could be reached bypolyadenylation of the mRNA. All mice treated with polyadenylated mRNAreached a level over potentially protective virus neutralizing titers(>40).

10. Vaccination with mRNA Encoding RAV G:

Balb/c mice were vaccinated 2 times (d0 and d21) with 20 μg RAV-G mRNAinto both M. tibialis. 8 animals were vaccinated i.m. with R2506 (SEQ IDNO: 15; naked RAV-G mRNA), 8 animals were vaccinated i.m. withpolyadenylated R2506 (naked RNA) and 8 mice were injected with RiLa ascontrols. Blood was collected 28 days after prime. Serum was analyzedfor VNTs.

10.1. Virus Neutralization Test

Detection of the virus neutralizing antibody response (specific B-cellimmune response) was carried out by a virus neutralisation assay. Theresult of that assay is referred to as virus neutralization titer (VNT).According to WHO standards, an antibody titer is considered protectiveif the respective VNT is at least 0.5 IU/ml. Therefore, blood sampleswere taken from vaccinated mice on day 28 and sera were prepared. Thesesera were used in fluorescent antibody virus neutralisation (FAVN) testusing the cell culture adapted challenge virus strain (CVS) of rabiesvirus as recommended by the OIE (World Organisation for Animal Health)and first described in Cliquet F., Aubert M. & Sagne L. (1998); J.Immunol. Methods, 212, 79-87. In brief, heat inactivated sera weretested as quadruplicates in serial two-fold dilutions with respect totheir potential to neutralise 100 TCID50 (tissue culture infectiousdoses 50%) of CVS in 50 μl of volume. Therefore, sera dilutions wereincubated with virus for 1 hour at 37° C. (in humid incubator with 5%CO2) and subsequently trypsinized BHK-21 cells were added (4×105cells/ml; 50 μl per well). Infected cell cultures were incubated for 48hours in humid incubator at 37° C. and 5% CO₂. Infection of cells wasanalysed after fixation of cells using 80% acetone at room temperatureusing FITC anti-rabies conjugate. Plates were washed twice using PBS andexcess of PBS was removed. Cell cultures were scored as positive or asnegative with regard to the presence of rabies virus. Negatively scoredcells in sera treated wells represent neutralization of rabies virus.Each FAVN tests included WHO or OIE standard serum (positive referenceserum) that served as reference for standardisation of the assay.Neutralization activity of test sera was calculated with reference tothe standard serum provided by the WHO and displayed as InternationalUnits/ml (IU/ml).

Results

As can be seen in FIG. 9, the polyadenylated RAV-G mRNA induces higherneutralizing antibody titers, well above the WHO standard of 0.5 IU/ml.

The invention claimed is:
 1. A method for increasing protein productionfrom a RNA molecule comprising providing the RNA molecule comprising: a)a 5′-cap structure; b) at least one open reading frame (ORF) encoding aprotein; and c) a heterologous 3′-untranslated region (3′-UTR)comprising at least a first and a second poly(A) sequence, wherein: (i)the first poly(A) sequence comprises at least 20 adenine nucleotides;and (ii) the second poly(A) sequence comprises at least 70 adeninenucleotides, wherein the first and the second poly(A) sequences areseparated by a nucleic acid sequence comprising from 10 to 90nucleotides and having no more than 2 consecutive adenine nucleotides,wherein the RNA molecule yields increased protein production whenexpressed in a cell or an organism in comparison to a reference nucleicacid molecule comprising an identical nucleic acid sequence as the RNAmolecule but lacking a second poly(A) sequence.
 2. The method of claim1, wherein at least one of the poly(A) sequences is located at the 3′terminus of the RNA molecule.
 3. The method of claim 1, wherein the RNAmolecule comprises a heterologous 5′ UTR sequence.
 4. The method ofclaim 1, wherein the protein is an antigen.
 5. The method of claim 4,wherein the antigen is an antigen from a bacterial, a viral, a fungal ora protozoan pathogen.
 6. The method of claim 4, wherein the antigen is aviral antigen.
 7. The method of claim 4, wherein the antigen is a tumorantigen.
 8. The method of claim 1, wherein the RNA molecule comprisesfrom 5′ to 3′: I) the 5′-cap structure; II) the heterologous 5′ UTRsequence; III) the at least one open reading frame (ORF) encoding aprotein; IV) the heterologous 3′-untranslated region (3′-UTR)comprising, from 5′ to 3′, (i) the first poly(A) sequence comprising atleast 20 adenine nucleotides; (ii) the nucleic acid sequence whichseparates the first and the second poly(A) sequence, comprising from 10to 90 nucleotides and having no more than 2 consecutive adeninenucleotides; and (iii) the second poly(A) sequence comprising at least70 adenine nucleotides.
 9. The method of claim 8, wherein the ORFencoding the protein has a G/C content that is increased relative to acorresponding reference ORF encoding the protein.
 10. The method ofclaim 9, wherein the ORF encoding the protein has a G/C content that isincreased by at least 7% relative to a corresponding reference ORFencoding the protein.
 11. The method of claim 10, wherein the ORFencoding the protein has a G/C content that is increased by at least 15%relative to a corresponding reference ORF encoding the protein.
 12. Themethod of claim 11, wherein the first poly(A) sequence comprises atleast 30 adenine nucleotides.
 13. The method of claim 12, wherein thenucleotide sequence which separates the first and the second poly(A)sequences consists of 10 nucleotides and has no more than 2 consecutiveadenine nucleotides.
 14. The method of claim 13, wherein the RNAmolecule comprises at least one nucleotide analog.
 15. The method ofclaim 14, wherein the at least one nucleotide analogue is a modifiedform of uridine.
 16. The method of claim 15, wherein the modified formof uridine is chemically altered by methylation.
 17. The method of claim16, wherein the modified form of uridine is a naturally occurringvariant of uridine.
 18. The method of claim 17, wherein the RNA moleculeis complexed with a cationic or polycationic compound.
 19. The method ofclaim 18, wherein the cationic or polycationic compound comprises acationic or polycationic peptide.
 20. The method of claim 18, whereinthe cationic or polycationic compound comprises a cationic lipid.
 21. Amethod for increasing protein production from a RNA molecule comprisingproviding the RNA molecule formulated in a pharmaceutical composition,where the RNA molecule comprises in the 5′ to 3′ direction: a) a 5′-capstructure; b) at least one open reading frame (ORF) encoding a proteinthat is a viral antigen; and c) a heterologous 3′-untranslated region(3′-UTR) comprising at least a first and a second poly(A) sequence,wherein: (i) the first poly(A) sequence comprises at least 20 adeninenucleotides; and (ii) the second poly(A) sequence comprises at least 70adenine nucleotides, wherein the first and the second poly(A) sequencesare separated by a nucleic acid sequence comprising from 10 to 90nucleotides and having no more than 2 consecutive adenine nucleotides,wherein the RNA molecule comprises at least one nucleotide analogue,which is a naturally occurring variant of uridine, wherein the RNAmolecule yields increased protein production when expressed in a cell oran organism in comparison to a reference nucleic acid moleculecomprising an identical nucleic acid sequence as the RNA molecule butlacking a second poly(A) sequence; wherein the ORF encoding the viralantigen has a G/C content that is increased by at least 15% relative toa corresponding reference ORF; and wherein the RNA molecule is complexedwith a cationic or polycationic compound comprising a cationic lipid.22. The method of claim 21, wherein the naturally occurring variant ofuridine is chemically altered by methylation.
 23. The method of claim21, wherein the RNA molecule is complexed with a cationic carrier or apolycationic carrier.
 24. The method of claim 23, wherein the cationicor polycationic compound comprises a cationic lipid.
 25. A method forincreasing protein production from a RNA molecule comprising providingthe RNA molecule formulated in a pharmaceutical composition, where theRNA molecule comprises: a) a 5′-cap structure; b) at least one openreading frame (ORF) encoding a protein; and c) a heterologous3′-untranslated region (3′-UTR) comprising at least a first and a secondpoly(A) sequence, wherein: (i) the first poly(A) sequence comprises atleast 20 adenine nucleotides; and (ii) the second poly(A) sequencecomprises at least 70 adenine nucleotides, wherein the first and thesecond poly(A) sequences are separated by a nucleic acid sequenceconsisting of 10 nucleotides and having no more than 2 consecutiveadenine nucleotides, wherein the ORF encoding the protein has a G/Ccontent that is increased by at least 15% relative to a correspondingreference ORF encoding the protein, wherein the RNA molecule yieldsincreased protein production when expressed in a cell or an organism incomparison to a reference nucleic acid molecule comprising an identicalnucleic acid sequence as the RNA molecule but lacking a second poly(A)sequence.
 26. The method of claim 25, wherein the ORF encoding theprotein is an antigen.
 27. The method of claim 26, wherein the antigenis a viral antigen.
 28. The method of claim 27, wherein the RNA moleculecomprises at least one nucleotide analogue, which is a naturallyoccurring variant of uridine.
 29. The method of claim 28, wherein theRNA molecule is complexed with a cationic carrier or a polycationiccarrier.
 30. The method of claim 29, wherein the cationic orpolycationic compound comprises a cationic lipid.